Nucleic acid molecules for reduction of papd5 or papd7 mrna for treating hepatitis b infection

ABSTRACT

The present invention relates to a method for identifying a compound that prevents, ameliorates and/or inhibits a hepatitis virus (HBV) infection, wherein a compound that reduces the expression and/or activity of PAP associated domain containing 5 (PAPD5) and/or PAP associated domain containing 7 (PAPD7) is identified as a compound that prevents, ameliorates and/or inhibits a BV infection. The invention also provides for inhibitors of PAPD5 or PAPD7 for use in treating and/or preventing a HBV infection; as well as a combined preparation comprising an inhibitor of PAPD5 and an inhibitor of PAPD7 for simultaneous or sequential use in the treatment or prevention of a HBV infection. Also comprised in the present invention is a pharmaceutical composition for use in the treatment and/or prevention of a HBV infection, and a method for monitoring the therapeutic success during the treatment of a HBV infection.

FIELD OF THE INVENTION

The present invention relates to a method for identifying a compoundthat prevents, ameliorates and/or inhibits a hepatitis B virus (HBV)infection, wherein a compound that reduces the expression and/oractivity of PAP associated domain containing 5 (PAPD5) or PAP associateddomain containing 7 (PAPD7) is identified as a compound that prevents,ameliorates and/or inhibits a HBV infection. The invention also providesfor inhibitors of PAPD5 or PAPD7 for use in treating and/or preventing aHBV infection. Specifically the present invention identifies nucleicacid molecules, such as antisense oligonucleotides or RNAi agents asinhibitors of PAPD5 or PAPD7 as well as a combined preparation of thesecomprising an inhibitor of PAPD5 and an inhibitor of PAPD7 forsimultaneous or sequential use in the treatment or prevention of a HBVinfection. Also comprised in the present invention is a pharmaceuticalcomposition for use in the treatment and/or prevention of a HBVinfection, and a method for monitoring the therapeutic success duringthe treatment of a HBV infection.

BACKGROUND

The hepatitis B virus (HBV) is an enveloped, partially double-strandedDNA virus. The compact 3.2 kb HBV genome consists of four overlappingopen reading frames (ORF), which encode for the core, polymerase (Pol),envelope and X-proteins. The Pol ORF is the longest and the envelope ORFis located within it, while the X and core ORFs overlap with the PolORF. The lifecycle of HBV has two main events: 1) generation of closedcircular DNA (cccDNA) from relaxed circular (RC DNA), and 2) reversetranscription of pregenomic RNA (pgRNA) to produce RC DNA. Prior to theinfection of host cells, the HBV genome exists within the virion as RCDNA. It has been determined that HBV virions are able to gain entry intohost cells by non-specifically binding to the negatively chargedproteoglycans present on the surface of human hepatocytes (Schulze,Hepatology, 46, (2007), 1759-68) and via the specific binding of HBVsurface antigens (HBsAg) to the hepatocyte sodium-taurocholatecotransporting polypeptide (NTCP) receptor (Yan, J Virol, 87, (2013),7977-91). The control of viral infection needs a tight surveillance ofthe host innate immune system which could respond within minutes tohours after infection to impact on the initial growth of the virus andlimit the development of a chronic and persistent infection. Despite theavailable current treatments based on IFN and nucleos(t)ide analogues,the HBV infection remains a major health problem worldwide whichconcerns an estimated 350 million chronic carriers who have a higherrisk of liver cirrhosis and hepatocellular carcinoma.

The secretion of antiviral cytokines in response to a HBV infection bythe hepatocytes and/or the intra-hepatic immune cells plays a centralrole in the viral clearance of the infected liver. However, chronicallyinfected patients only display a weak immune response due to variousescape strategies adopted by the virus to counteract the host cellrecognition systems and the subsequent antiviral responses.

Many observations showed that several HBV viral proteins couldcounteract the initial host cellular response by interfering with theviral recognition signaling system and subsequently the interferon (IFN)antiviral activity. Among these, the excessive secretion of HBV emptysubviral particles (SVPs, HBsAg) are thought to participate to themaintenance of the immunological tolerant state observed in chronicallyinfected patients (CHB). The persistent exposure to HBsAg and otherviral antigens can lead to HBV-specific T-cell deletion or toprogressive functional impairment (Kondo, Journal of Immunology (1993),150, 4659-4671; Kondo, Journal of Medical Virology (2004), 74, 425-433;Fisicaro, Gastroenterology, (2010), 138, 682-93;). Moreover HBsAg hasbeen reported to suppress the function of immune cells such asmonocytes, dendritic cells (DCs) and natural killer (NK) cells by directinteraction (Op den Brouw, Immunology, (2009b), 126, 280-9; Woltman,PLoS One, (2011), 6, e15324; Shi, J Viral Hepat. (2012), 19, e26-33;Kondo, ISRN Gasteroenterology, (2013), Article ID 935295).

HBsAg quantification is a significant biomarker for prognosis andtreatment response in chronic hepatitis B. However the achievement ofHBsAg loss and seroconversion is rarely observed in chronically infectedpatients but remains one of the ultimate goals of therapy. Currenttherapy such as Nucleos(t)ide analogues are molecules that inhibit HBVDNA synthesis but are not directed at reducing HBsAg level.Nucleos(t)ide analogs, even with prolonged therapy, only show weak HBsAgclearance comparable to those observed naturally (between −1%-2%)(Janssen, Lancet, (2005), 365, 123-9; Marcellin, N. Engl. J. Med.,(2004), 351, 1206-17; Buster, Hepatology, (2007), 46, 388-94).

Hepatitis B e-antigen (also called HBV envelope antigen or HBeAg) is aviral protein that is secreted by hepatitis B infected cells. HBeAg isassociated with chronic hepatitis B infections and is used as a markerof active viral disease and a patient's degree of infectiousness.

The function of the hepatitis B virus precore or HBeAg is not completelyknown. However HBeAg is well known to play a key role in viralpersistence. HBeAg is thought to promote HBV chronicity by functioningas an immunoregulatory protein. In particular, the HBeAg is a secretedaccessory protein, which appears to attenuate the host immune responseto the intracellular nucleocapsid protein (Walsh, Virology, 2011,411(1):132-141). The HBeAg acts as an immune tolerogen contributing toHBV persistence, and possibly functions in utero considering thatsoluble HBeAg traverses the placenta (Walsh, Virology, 2011,411(1):132-141). Furthermore, HBeAg downregulates: i) cellular genescontrolling intracellular signaling; and ii) the Toll-like receptor 2(TLR-2) to dampen the innate immune response to viral infection (Walsh,Virology, 2011, 411(1):132-141). In the absence of HBeAg, HBVreplication is associated with upregulation of the TLR2 pathway (Walsh,Virology, 2011, 411(1):132-141). Accordingly, HBeAg has a significantrole in modulating virus/host interactions to influence the host immuneresponse (Walsh, Virology, 2011, 411(1):132-141). Thus, reducing HBeAgin HBeAg positive patient population may lead to reversal of HBVspecific immunedysfunction (Milich, 1997, J. Viral. Hep. 4: 48-59;Milich, 1998, J. Immunol. 160: 2013-2021). In addition, the secretedHBeAg is significantly more efficient than the intracellular hepatitiscore antigen (HBcAg) at eliciting T-cell tolerance, and the split T-celltolerance between the HBeAg and the HBcAg and the clonal heterogeneityof HBc/HBeAg-specific T-cell tolerance may have significant implicationsfor natural HBV infection and especially for precore-negative chronichepatitis (Chen, 2005, Journal of Virology, 79: 3016-3027).

Accordingly, reducing secretion of HBeAg in addition to secretion ofHBsAg would lead to an improved inhibition of development of a chronicHBV infection as compared to the inhibition of secretion of HBsAg alone.In addition, the highest rates of transmission of an acute infection tochronic (>80%) have been reported in cases of materno-fetal and neonatalHBV transmission from HBeAg-positive mothers (Liaw, Lancet, 2009, 373:582-592; Liaw, Dig. Dis. Sci., 2010, 55: 2727-2734; and Hadziyannis,2011, Journal of hepatology, 55: 183-191). Therefore, reducing HBeAg inan expected mother may not only reduce the patient's degree ofinfectiousness, but may also inhibit the development of a chronic HBVinfection of her child.

Therefore, in the therapy of HBV there is an unmet medical need toinhibit viral expression, particularly to inhibit secretion of HBsAg andHBeAg (Wieland, S. F. & F. V. Chisari. J Virol, (2005), 79, 9369-80;Kumar et al. J Virol, (2011), 85, 987-95; Woltman et al. PLoS One,(2011), 6, e15324; Op den Brouw et al. Immunology, (2009b), 126, 280-9).

WO 03/022987 discloses for example in Table 7A 1298 genes that areupregulated in hepatitis C-positive tissue. One of the mentioned genesis topoisomerase-related function protein 4 (TRF4, AF089897). AF089897is also called TRF4-2, which is quite similar to position 880 to 2340 ofSEQ ID NO: 4 herein. The observation that a fragment of PAPD5 isupregulated slightly in hepatitis C positive cells does not provide anyindication that inhibiting PAPD5 represents an effective therapy. WO03/022987A2 does not disclose any hint that fragments of PAPD5 plays anycritical role during hepatitis C infection at all. In addition, HCV andHBV are two completely different viruses leading to two completelydifferent diseases with different etiologies, different progression anddifferent medication. This is in line with the observation of thepresent inventors that the PAPD5 and PAPD7 inhibitors DHQ and THP areinactive against hepatitis C virus (HCV) or other viruses beside HBV(data not shown).

In WO 2010/040571 PAPD5 has been suggested in a long list of other genesas having a potential role in cell proliferation in metabolic andtumorous disease without the provision of any actual evidence.

In WO 2013/166264 PAPD5 has been suggested in a long list of other genesas having a potential role in increasing viral replication without theprovision of any actual evidence.

In WO 2017/066712 down regulation of PAPD5 in relation to the treatmentand diagnosis of telomere diseases has been described. Five shRNAstructures for this purpose have been described.

To our knowledge the expression of PAPD5 or PAPD7 has never beenassociated with HBV infection, nor has modified single strandedantisense oligonucleotides been made against these targets.

OBJECTIVE OF THE INVENTION

Thus, the technical problem underlying the present invention is theidentification and provision of ameliorated means and methods fortreating and/or preventing a HBV infection.

The technical problem is solved by the provision of the embodimentsdescribed herein and characterized in the claims.

BRIEF DESCRIPTION OF THE FIGURES

The Figures show:

FIG. 1 : Pictures from 1-by-1 experiment with HBX129653/HBX129654chemical probes and the 3 prey fragments.

FIG. 2 : Pictures from 1-by-1 experiment with HBX129653/HBX129654chemical probes and PAPD5/7 full length proteins.

FIG. 3 : Pictures from competition assay using HBX129653 (DHQ) andMOL653/654 for competition

FIG. 4 : Pictures from competition assay using HBX129654 (THP) andMOL653/654 for competition

FIG. 5 : Pictures from competition assay using HBX129653 (DHQ) andINACT653/INACT654 for competition. MOL653 was included as positivecontrol.

FIG. 6 : Pictures from competition assay using HBX129654 (THP) andINACT653/INACT654 for competition. MOL653 was included as positivecontrol.

FIG. 7 : (A) SiRNA knock-down (KD) of PAPD5 and PAPD7 in HBV-infecteddHepaRG leads to reduction in HBV expression. Differentiated HepaRGcells were infected with HBV and treated with siRNA against eitherPAPD5, PAPD7 or both (25 nM each) one day prior to HBV infection and onday 4 post infection. Supernatant were harvested on day 11, levels ofHBsAg and HBeAg secreted in the supernatant were measured by ELISA andnormalized to non-treated control. Cell toxicity and inhibition of geneexpression was measured subsequently and also normalized to thenon-treated control. (B) The same experiment as described in (A) wasperformed, with the exception that only the level of HBsAg secreted inthe supernatant was measured.

FIG. 8 : Representation of the ability of the oligonucleotides, testedin example 4, to reduce the expression of PAPD5 in HeLa cell cultures.Each oligonucleotide is represented by a dot indicating its location onthe PAPD5 mRNA by chromosomal position. The oligonucleotideconcentrations were 5 and 25 microM as indicated in the right hand sideeach plot.

FIG. 9 : Representation of the ability of the oligonucleotides, testedin example 5, to reduce the expression of PAPD7 in HeLa cell cultures.Each oligonucleotide is represented by a dot indicating its location onthe PAPD7 mRNA by chromosomal position. The oligonucleotideconcentrations were 5 and 25 microM as indicated in the right hand sideeach plot.

FIG. 10 : Representation of the inhibition of HBsAg in HBV infectedHepaRG cells with combinations oligonucleotides targeting PAPD5 andPAPD7 (20 μM each) compared to the inhibition obtained using a singleoligonucleotide (20 μM) present in the combination. The assay wasconducted with gymnosis.

FIG. 11 : Representation of the inhibition of HBsAg in HBV infectedHepaRG cells with combinations oligonucleotides targeting PAPD5 andPAPD7 (500 nM each) compared to the inhibition obtained using a singleoligonucleotide (500 nM) from the combination. The assay was conductedwith transfection.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a composition comprisinga nucleic acid molecule for use in the treatment and/or prevention ofHepatitis B virus infection, wherein said nucleic acid molecule inhibitsexpression and/or activity of PAPD5. In particular a compositioncomprising a combined preparation of a nucleic acid molecule inhibitsexpression and/or activity of PAPD5 and another nucleic acid moleculeinhibits expression and/or activity of PAPD7 for use in the treatmentand/or prevention of Hepatitis B virus infection.

A further aspect of the present invention relates to nucleic acidmolecules that inhibit expression and/or activity of PAPD7. Inparticular single stranded antisense, siRNA and shRNA molecules.

A further aspect of the present invention relate to single strandedantisense oligonucleotides that inhibit expression and/or activity ofPAPD5. In particular modified antisense oligonucleotides comprising2′sugar modified oligonucleotide and phosphorothioate internucleosidelinkages.

Further aspects of the invention are conjugates of nucleic acidmolecules of the invention, combined preparations of nucleic acidmolecules capable of inhibiting expression and/or activity of both PAPD5and PAPD7 and pharmaceutical compositions comprising the molecules ofthe invention.

A further aspect of the invention is a method for identifying a compoundor composition that prevents, ameliorates and/or inhibits a hepatitis Bvirus (HBV) infection, comprising:

-   -   a. contacting a test compound or composition with a cell        expressing PAPD5 and/or PAPD7;    -   b. measuring the expression and/or activity of PAPD5 and/or        PAPD7 in the presence and absence of said test compound or        composition; and    -   c. identifying a compound or composition that reduces the        expression and/or activity of PAPD5 and/or PAPD7 as a compound        that prevents, ameliorates and/or inhibits a HBV infection.

DETAILED DESCRIPTION OF THE INVENTION

PAPD5 and PAPD7 are non-canonical poly(A)-polymerases that belong to thesuperfamily of polymerase β-like nucleotidyl transferases. In context ofthe present invention it has surprisingly been shown that a compoundthat is useful for the therapeutic intervention of a HBV infection cansuccessfully be identified by analysing whether a test compound inhibitsPAPD5 or PAPD7. Or, in other words, inhibition of PAPD5 or PAPD7, or theinhibition of both, was identified in the appended examples as being anindicator for the efficacy of a compound to inhibit a HBV infection. Theappended examples demonstrate that a dihydroquinolizinone compoundhaving the formula (III) shown in the Materials and Methods section,herein called DHQ, and a tetrahydropyridopyrimidine compound having theformula (IV) as shown in the Materials and Methods section,herein calledTHP, bind to PAPD5 and PAPD7 polypeptides (SEQ ID NO: 1 and 2respectively). These compounds have the capacity to inhibit productionof HBV surface antigen (HBsAg) and the expression of HBV RNA during HBVinfection (WO 2015/113990 A1 and WO2016/177655). In addition, theappended examples show that inhibition of PAPD5 or PAPD7 or both byusing pools of siRNA leads to an inhibition of viral expression,particularly of the secretion of HBsAg and HBeAg as well as of theproduction of intracellular HBV mRNA. These results directly indicatethat by reducing the amount and/or activity (e.g. the amount) of PAPD5and/or PAPD7 an HBV infection (e.g. a chronic HBV infection) can beprevented or treated (i.e. ameliorated and/or inhibited).

Screening Methods of the Invention

Thus, the present invention relates to a screening method, wherein acompound that reduces the expression and/or activity (e.g. theexpression) of PAPD5 or PAPD7 (or combinations of compounds thatreducePAPD5 and PAPD7) is identified as a compound that prevents and/ortreats (i.e. ameliorates and/or inhibits) a HBV infection. In apreferred embodiment of the present invention the compound is a RNAimolecule, in particular a nucleic acid molecule, such as a siRNA, shRNAor antisense oligonucleotide. Using the screening method of theinvention 240 LNA modified antisense oligonucleotides targeting eitherPAPD5 or PAPD7 mRNA have been screened for their ability to reduce theexpression of PAPD5 or PAPD7, or both using combinations of compounds.Some of these have further been tested to confirm their ability toameliorate and/or inhibits a HBV infection, either alone or incombination.

One aspect of the invention is a method for identifying a compound orcomposition that prevents, ameliorates and/or inhibits a hepatitis Bvirus (HBV) infection, comprising:

-   -   a) contacting a test compound with a cell expressing PAP        associated domain containing 5 (PAPD5) and/or PAP associated        domain containing 7 (PAPD7);    -   b) measuring the expression and/or activity of PAPD5 and/or        PAPD7 in the presence and absence of said test compound or        composition; and    -   c) identifying a compound that reduces the expression and/or        activity of PAPD5 or PAPD7 as a compound or composition that        prevents, ameliorates and/or inhibits a HBV infection;        optionally    -   d) testing combinations of compounds to reduce the expression        and/or activity of PAPD5 and PAPD7.

It has been found in context of the present invention that a compound(or composition) that reduces PAPD5 or PAPD7 or combinations ofcompounds that reduce PAPD5 and PAPD7 in combination leads to inhibitionof HBV gene expression and replication; and thus, prevents, amelioratesand/or inhibits a HBV infection. Such a compound may lead to a reductionof the PAPD5 or PAPD7 expression and/or activity of 10-100%, preferablyof 20-100%, more preferably of 30-100%, even more preferably of 40-100%,even more preferable of 50-100%, even more preferably of 60-100%, evenmore preferably of 70-100%, even more preferably of 80-100%, and mostpreferably of 90-100%.

In the herein provided screening method it is envisaged that theexpression of PAPD5 and/or PAPD7 is measured (i.e. analyzed/determined)by using in step (a) a cell expressing PAPD5 and/or PAPD7, such as aHeLa or a HepaRG cell line. The expression and/or activity of PAPD5and/or PAPD7 may be measured (i.e. analyzed/determined) by either (i)determining PAPD5 and/or PAPD7 polypeptide; or (ii) determiningtranscript levels in a cell expressing PAPD5 and/or PAPD7.

In one aspect of the invention, a compound that reduces the expressionof PAPD5 or PAPD7 (e.g. of PAPD5, or preferably combinations ofcompounds that reduce both PAPD5 and PAPD7) is identified as acompound(s) that prevents, ameliorates and/or inhibits (i.e. treats) HBVinfection. In another aspect of the invention a compound that reducesthe activity of PAPD5 or PAPD7 polypeptide (e.g. of PAPD5, or preferablycombinations of compounds that reduce both PAPD5 and PAPD7) isidentified as a compound(s) that prevents, ameliorates and/or inhibits(i.e. treats) a HBV infection. It is prioritized that a compound thatreduces the expression and/or activity of PAPD5 or combinations ofcompounds that reduce both molecules, PAPD5 and PAPD7, is identified ascompounds that prevents, ameliorates and/or inhibits a HBV infection.Most preferably, a combination of compounds that reduces the expressionand/or activity of both molecules, PAPD5 and PAPD7, is identified as acomposition that prevents, ameliorates and/or inhibits a HBV infection.

The above described screening method lead to the identification of acompound or combination of compounds, that prevents, ameliorates and/orinhibits a HBV infection. It is prioritized that said compoundsameliorates and/or inhibits (i.e. treats) a HBV infection. Thus, theherein provided screening method is useful in the identification of acompound that treats a HBV infection.

In the context of the present invention, PAPD5 may be the PAPD5polypeptide or the PAPD5 mRNA. It is prioritized in context of thescreening methods provided herein that PAPD5 is the PAPD5 mRNA.

One aspect of the present invention relates to the herein providedscreening method, wherein the cells expressing PAPD5 contain a PAPD5target nucleic acid comprising or consisting of

(i) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1 or 2;

(ii) a nucleotide sequence of SEQ ID NO: 4, 5 or 10 or natural variantsthereof;

(iii) a nucleotide sequence encoding an amino acid sequence having atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 98%, and even more preferably at least 99%identity to SEQ ID NO: 1 or 2, wherein the polynucleotide encodes apolypeptide that has poly-A polymerase function;

(iv) a nucleotide sequence having at least 80% identity, preferably atleast 90%, more preferably at least 95%, even more preferably at least98%, and even more preferably at least 99% identity to the nucleotidesequence of (ii), wherein the polypeptide expressed from the nucleotidesequence has poly-A polymerase function;

(v) a nucleotide sequence encoding an enzymatically active fragment ofSEQ ID NO: 1 or 2, such as a nucleotide sequence encoding SEQ ID NO: 7or 8;

(vi) a nucleotide sequence encoding an amino acid sequence having atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 98%, and even more preferably at least 99%identity to an amino acid sequence of an enzymatically active fragmentof SEQ ID NO: 1 or 2, such as SEQ ID NO: 7 or 8, wherein thepolynucleotide encodes a polypeptide that has poly-A polymerasefunction; or

(vii) a nucleotide sequence comprising or consisting of SEQ ID NO: 4, 5or 10.

In preferred embodiments, the PAPD5 target nucleic acid is a mRNA, suchas a pre-mRNA or mature mRNA. In further embodiments the PAPD5 targetnucleic acid is a polynucleotide comprising or consisting of thenucleotide sequence of SEQ ID NO: 4, 5 or 10 or natural variantsthereof. However, the PAPD5 mRNA may also be a polynucleotide comprisingor consisting of a nucleotide sequence having at least 80%, preferablyat least 90%, more preferably at least 95%, even more preferably atleast 98%, and even more preferably at least 99% identity to SEQ ID NO:4, 5 or 10, wherein the polynucleotide encodes a polypeptide that haspoly-A polymerase function.

In context of the present invention PAPD7 may be the PAPD7 polypeptideor the PAPD7 mRNA. It is prioritized in context of the screening methodsprovided herein that PAPD7 is the PAPD7 mRNA.

One aspect of the present invention relates to the herein providedscreening methods, wherein the cells expressing PAPD7 contain a PAPD7target nucleic acid comprising or consisting of

(i) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3;

(ii) a nucleotide sequence of SEQ ID NO: 6 or 11 or natural variantsthereof;

(iii) a nucleotide sequence encoding an amino acid sequence having atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 98%, and even more preferably at least 99%identity to SEQ ID NO: 3, wherein the polynucleotide encodes apolypeptide that has poly-A polymerase function;

(iv) a nucleotide sequence having at least 80% identity, preferably atleast 90%, more preferably at least 95%, even more preferably at least98%, and even more preferably at least 99% identity to an nucleic acidsequence of (ii), wherein the polypeptide expressed from the nucleicacid sequence has poly-A polymerase function;

(v) a nucleotide sequence encoding an enzymatically active fragment ofSEQ ID NO: 3, such as a nucleotide sequence encoding SEQ ID NO: 9; or

(vi) a nucleotide sequence encoding an amino acid sequence having atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 98%, and even more preferably at least 99%identity to an amino acid sequence of an enzymatically active fragmentof SEQ ID NO: 3, such as SEQ ID NO: 9, wherein the polynucleotideencodes a polypeptide that has poly-A polymerase function; or

(vii) a nucleotide sequence comprising or consisting of SEQ ID NO: 6 or11.

In preferred embodiments, the PAPD7 target nucleic acid is a mRNA, suchas a pre-mRNA or mature mRNA. In further embodiments the PAPD7 targetnucleic acid is a polynucleotide comprising or consisting of thenucleotide sequence of SEQ ID NO: 6 or 11, or natural variants thereof.However, the PAPD7 mRNA may also be a polynucleotide comprising orconsisting of a nucleotide sequence having at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least98%, and even more preferably at least 99% identity to SEQ ID NO: 6 or11, wherein the polynucleotide encodes a polypeptide that has poly-Apolymerase function.

In context of the present invention the cell used for screening may be aeukaryotic cell. For example, said cell may be a yeast cell or avertebrate cell. Vertebrate cells include fish, avian, reptilian,amphibian, marsupial, and mammalian cells. Preferably, the cell is amammalian cell, most preferably, a human cell. Mammalian cells alsoinclude feline, canine, bovine, equine, caprine, ovine, porcine murine,such as mice and rat, and rabbit cells. In the herein provided screeningmethods, the “cell” may endogenously express PAPD5 and/or PAPD7 oroverexpress PAPD5 and/or PAPD7. For overexpressing PAPD5 and/or PAPD7the cell may comprise the nucleotide sequence encoding the PAPD5polypeptide and/or the PAPD7 polypeptide within an expression vector. Inpreferred embodiments the cell comprises a nucleotide sequence encodingthe PAPD5 polypeptide and a nucleotide sequence encoding the PAPD7polypeptide. The cell of the herein provided screening methods may becomprised in a non-human animal, e.g. a mouse, rat, rabbit or ferret.The cells provided for the screening method described herein may also betermed target cells.

In the herein provided screening method wherein the activity of PAPD5polypeptide and/or PAPD7 polypeptide is measured, said activity of PAPD5and PAPD7 is preferably the poly-A polymerase function (i.e. the poly-Apolymerase activity). The poly-A polymerase function/activity of apolypeptide (e.g. of PAPD5 or PAPD7) may be measured, e.g. by monitoringthe in vitro polyadenylation of mRNA, e.g. as described in Rammelt, RNA,2011, 17:1737-1746. This method can also be used to measure the poly-Apolymerase function of PAPD5 and/or PAPD7 in the presence and absence ofa test compound. In brief, a ribo-oligonucleotide A₁₅ may be incubatedwith recombinant PAPD5 protein expressed in Escherichia coli in thepresence of ATP(A), CTP (C), GTP(G), UTP(U), or a mixture of all fourdNTPs, respectively.

The expression of PAPD5 and/or PAPD7 in a cell in the presence andabsence of the test compound may be measured, e.g. by using (q)PCR,western blot, or MassSpec.

A compound that inhibits the propagation of HBV may be a compound thatreduces the expression of viral RNA, that reduces the production ofviral DNA (HBV DNA) derived from viral RNA (HBV RNA), that reduces theproduction of new viral particles (HBV particles), and/or that producesproduction and/or secretion of HBsAg and/or HBeAg. Thus, one aspect ofthe present invention relates to the herein provided screening methods,wherein the compound that inhibits propagation of HBV inhibits secretionof HBsAg, inhibits secretion of HBeAg, and/or inhibits production ofintracellular HBV mRNA or HBV DNA. Preferably, a compound that inhibitsthe propagation of HBV is a compound that inhibits secretion of HBsAg,secretion of HBeAg and production of intracellular HBV mRNA or HBV DNA.

For example, a compound that inhibits propagation of HBV may reduce theexpression of viral RNA (HBV RNA), the production of viral DNA (HBV DNA)deriving from viral RNA, the production of new viral particles (HBVparticles), the production and/or secretion of HBsAg and/or HBeAg by10-100%, preferably by 20-100%, more preferably by 30-100%, even morepreferably by 40-100%, even more preferable by 50-100%, even morepreferably by 60-100%, even more preferably by 70-100%, even morepreferably by 80-100%, and most preferably by 90-100%, when compared theuntreated cells or animals or cell or animal treated with an appropriatecontrol.

Inhibition of propagation of HBV may be measured, e.g., by measuringwhether the test compound has the activity to inhibit secretion of HBsAgand/or of HBeAg, and/or to inhibit production of intracellular HBV mRNAor HBV DNA. Inhibition of secretion of HBsAg and/or HBeAg may bemeasured by ELISA, e.g. by using the CLIA ELISA Kit (Autobio Diagnostic)according to the manufacturers' instructions. Inhibition of productionof intracellular HBV mRNA may be measured by real-time PCR, e.g. asdescribed in the appended examples. Further methods for evaluatingwhether a test compound inhibits propagation of HBV are measuringsecretion of HBV DNA by RT-qPCR e.g. as described in WO 2015/173208 oras described in the appended examples; Northern Blot; in-situhybridization, or immuno-fluorescence.

The herein provided screening methods may additionally comprise the stepof comparing the test compound to a control. Said control may be aninactive test compound, wherein said inactive test compound is acompound that does not reduce the expression and/or activity of PAPD5 orPAPD7.

This inactive test compound has no activity against HBV, e.g. it doesnot lead to inhibition of secretion of HBsAg and HBeAg and to inhibitionof production of intracellular HBV mRNA. For example, the inactive testcompound may have an IC₅₀ value in the inhibition of HBsAg of more than6 μM. In the herein provided screening method, the inactive testcompound may be a non-targeting antisense oligonucleotide, siRNA orshRNA. In the screening method wherein expression and/or activity ofPAPD5 and/or PAPD7 is measured, the test compound as defined above in(i) may be used. An inactive compound can be designed from an activeone, e.g., by chemical modification and/or functional interruption.

For performing the herein provided screening methods publicly orcommercially available molecule libraries may be used. Thus, in contextof the invention the said test compound may be a screening library ofnucleic acid molecules selected from

(i) single stranded antisense oligonucleotides, preferably comprising atleast on 2′ modified nucleoside; or

(ii) siRNA molecules; or

(iii) shRNA molecules.

The appended examples demonstrate that by inhibiting PAPD5 and/or PDPD7polypeptide or mRNA, the secretion of HBsAg and HBeAg as well asproduction of intracellular HBV mRNA can effectively be inhibited. Thesedata demonstrate that an inhibitor of PAPD5 and/or PAPD7 can be used toprevent and/or treat a HBV infection.

Several small molecule compounds that have a certain efficacy in thetreatment of a HBV infection have been described in the art (see, e.g.WO 2015/113990 A1 and WO 2016/177655). The appended examples demonstratefor the first time a clear correlation between activity of the smallmolecule compound against a HBV infection and binding affinity towardsPAPD5 and PAPD7. This realization opened for design of nucleic acidmolecules targeting PAPD5 or PAPD7 mRNA leading to particularly highanti-HBV efficacy. The nucleic acid molecules can be targeted directlyto the liver using conjugates capable of binding to theasialoglycoprotein receptor (ASGPr). Compared to systemicallyadministered small molecules the nucleic acid molecules will have adifferent PK/PD profile and toxicity profile. Furthermore, the presentinvention shows for the first time that a compound(s) that inhibitsPAPD5 or PAPD7, or particularly PAPD5 and PAPD7 has an extraordinaryhigh activity in terms of inhibition of secretion of HBsAg and HBeAg aswell as of production of intracellular HBV mRNA. Reduction of secretionof HBsAg and HBeAg inhibits development of chronic HBV infection moreeffectively as compared to the reduction of secretion of HBsAg alone. Inaddition, inhibition of secretion of HBsAg and HBeAg reduces theinfectiousness of a HBV infected person. Furthermore, reducing HBeAg inan expected mother may also inhibit the development of a chronic HBVinfection of her child. Thus, the present invention unexpectedlydemonstrates that selectively using compounds that inhibit PAPD5 orPAPD7 target nucleic acids or combinations of compounds that inhibitboth PAPD5 and PAPD7 target nucleic acids, leads to an improvedtherapeutic success in the treatment of a HBV infection in terms of aconsiderably more effective reduction of HBsAg and HBeAg.

Accordingly, an aspect of the present invention is using one or moreinhibitors capable of reducing PAPD5 or PAPD7 target nucleic acids orcombinations of compounds that inhibit expression of both PAPD5 andPAPD7 target nucleic acids, in the treatment of HBV infection, inparticular a chronic HBV infection. In a further embodiment theinvention relates to the use of at least two inhibitors capable ofreducing PAPD5 and/or PAPD7 target nucleic acids, in reduction of theviral antigens HBsAg and HBeAg.

Thus, the present invention relates to an inhibitor or a combination ofinhibitors of PAPD5 and/or PAPD7 for use in treating and/or preventing aHBV infection, wherein said inhibitor(s) are independently selected fromthe group consisting of:

(a) one or more RNA interference (RNAi) molecules against PAPD5 orPAPD7;

(b) a genome editing machinery, comprising:

-   -   (i) a site-specific DNA nuclease or a polynucleotide encoding a        site-specific DNA nuclease; and    -   (ii) a guide RNA or a polynucleotide encoding a guide RNA.

The RNAi molecules may independently be selected from the groupconsisting of:

a) a single stranded antisense oligonucleotide;

b) a siRNA molecule; and

c) a shRNA molecule;

The inhibitor of the present invention may also be a PAPD5 or PAPD7specific locked nucleic acid (LNA) molecule.

It is envisaged that the inhibitor of the invention is used for treating(e.g. ameliorating) a HBV infection.

The inhibitor may be a molecule that specifically inhibits PAPD7.Preferably, the inhibitor is a molecule that specifically inhibitsPAPD5. More preferably, the inhibitors are combined such that theyinhibit both, PAPD5 and PAPD7. Thus, it is prioritized that theinhibitors of the present invention either inhibits PAPD5 or arecombined such that they inhibit both PAPD5 and PAPD7. Most preferably,the inhibitors of the present invention are combined such that theyinhibit PAPD5 and PAPD7. In one aspect of the invention the inhibitorsof the present invention are combined such that they inhibit both PAPD5and PAPD7 and lead to a reduction of secretion of HBsAg and/or HBeAg ofat least 50% as compared to the no drug control (i.e. compared to cellsor subjects to which no drug has been administrated).

The inhibitor of the present invention may have an 10₅₀ value in theinhibition of HBsAg and HBeAg of below 6 μM, preferably of below 5 μM,preferably of below 4 μM, preferably of below 3 μM, preferably of below2 μM, more preferably below 1 μM, more preferably below 0.5 μM, and mostpreferably below 0.1 μM.

Genome editing by using a site-specific DNA nuclease (such as Cas9 orCpf1) and a guide RNA is commonly known in the art and described, e.g.,in “CRISPR-Cas: A Laboratory Manual”, 2016, edited by Jennifer Doudna,ISBN 978-1-621821-31-1.

For example, if said site-specific DNA nuclease is a Cas9 nuclease, thenthe genome editing machinery preferably further comprises:

(i) at least one guide IRNA consisting of at least one target sequencespecific CRISPR IRNA (crRNA) molecule and at least one trans-activatingcrRNA (tracrRNA) molecule;

(ii) a polynucleotide encoding the RNA molecules of (i);

(iii) at least one guide RNA, which is a chimeric RNA moleculecomprising at least one target sequence specific crRNA and at least onetracrRNA; or

(iv) a polynucleotide encoding the chimeric RNA of (iii).

In an alternative example the site-specific DNA nuclease is a Cpf1nuclease, and the genome editing machinery preferably further comprises:

(i) at least one guide RNA comprising a target sequence specific CRISPRRNA (crRNA) molecule; or

(ii) a polynucleotide encoding the RNA molecules of (i).

The herein provided inhibitors of PAPD5 or PAPD7 may also be a genomeediting machinery that comprises at least one pre-assembled Cas9protein-guide RNA ribonucleoprotein complex (RNP).

Herein, the guide RNA is designed to target the genomic PAPD5 or PAPD7DNA. Alternatively, several guide RNAs are used, so that the genomic DNAof PAPD5 and of PAPD7 can be targeted. Inhibition of PAPD5 and/or PAPD7may be achieved by introducing frame-shift knockout mutations into thegenomic PAPD5 and/or PAPD7 DNA through non-homologous end-joining(NHEJ), or by modifying the genomic PAPD5 and/or PAPD7 DNA throughhomology-directed repair (HDR). How these mechanisms can be induced iscommonly known in the art and described, e.g., in Heidenreich, 2016, NatRev Neurosci 17 36-44.

The inhibitor of the present invention of the present invention ispreferably a non-naturally occurring molecule. The inhibitor of theinvention may be a nucleic acid molecule, selected from RNAi agents,including siRNA, shRNA, Crisper RNA and single stranded antisenseoligonucleotides. Preferably the RNAi molecules comprise at least onenon-naturally occurring nucleotide, such as a oligonucleotidethiophosphate, a substituted ribo-oligonucleotide, a 2′ sugar modifiednucleoside, a LNA nucleoside, a PNA nucleoside, a GNA (glycol nucleicacid) molecule, a TNA (threose nucleic acid) molecule, a morpholinonucleotide, or a nucleic acid with a modified backbone such aspolysiloxane, 2′-O-(2-methoxy) ethyl-phosphorothioate, or a nucleic acidwith a substituent, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,amino-, propyl-, chloro-, and methanocarbanucleoside, or a reportermolecule to facilitate its detection. The inhibitor of the invention mayalso be naturally occurring or a non-naturally occurring small moleculeor genome editing machinery.

In context of the present invention, the herein provided inhibitorinhibits expression and/or activity of PAPD5 or PAPD7.

For example, the inhibitor of the present invention may bind to PAPD5target nucleic acid and inhibit activity of PAPD5 polypeptide. Inanother example, the inhibitor of the present invention binds to PAPD7target nucleic acid and inhibits activity of PAPD7 polypeptide. It isprioritized herein that the inhibitors are combined to target both,PAPD5 and PAPD7 mRNA and inhibits the activity of both, PAPD5 and PAPD7polypeptide. The inhibitor of the present invention may inhibit theexpression of PAPD5 or PAPD7; or a combination of inhibitors may inhibitthe expression of both, PAPD5 and PAPD7.

Compounds of the Invention

As described above, the inhibitor of the present invention may be anucleic acid molecule.

In one aspect of the invention, the inhibitor of the present is a RNAimolecule against PAPD5 or PAPD7. Said RNAi molecule may be a siRNA or ashRNA.

For example, the inhibitor of the present invention may be a siRNA thatis directed against PAPD5, wherein said siRNA is any one of or acombination of the following siRNAs:

PAPD5 siRNA Pool (L-010011-00-0010; ON-TARGETplus Human PAPD5):

siRNA-1-J-010011-05-Target Sequence: (SEQ ID NO: 252)CAUCAAUGCUUUAUAUCGA siRNA-2-J-010011-06-Target Sequence:(SEQ ID NO: 253) GGACGACACUUCAAUUAUUsiRNA-3-J-010011-07-Target Sequence: (SEQ ID NO: 254)GAUAAAGGAUGGUGGUUCA siRNA-4-J-010011-08-Target Sequence:(SEQ ID NO: 255) GAAUAGACCUGAGCCUUCA

The inhibitor of the present invention may also be a siRNA that isdirected against PAPD7, wherein said siRNA is any one of or acombination of the following siRNAs:

PAPD7 siRNA Pool (L-009807-00-0005; ON-TARGETplus Human PAPD7):

siRNA-1-J-009807-05-Target Sequence: (SEQ ID NO: 256)GGAGUGACGUUGAUUCAGA siRNA-2-J-009807-06-Target Sequence:(SEQ ID NO: 257) CGGAGUUCAUCAAGAAUUAsiRNA-3-J-009807-07-Target Sequence: (SEQ ID NO: 258)CGGAGUUCAUCAAGAAUUA siRNA-4-J-009807-08-Target Sequence:(SEQ ID NO: 259) GCGAAUAGCCACAUGCAAU

Above, target sequences of suitable siRNAs are shown. The sequences ofthe corresponding siRNAs are directly complementary to these targetsequences.

It is envisaged in context of the present invention that a combinedpreparation may comprise (a) siRNA(s) directed against PAPD5 is combinedwith (b) siRNA(s) directed against PAPD7, in order to inhibit expressionof both, PAPD5 and PAPD7.

It is also envisaged in context of the present invention that a combinedpreparation may comprise (a) shRNA directed against PAPD5 is combinedwith (b) shRNA directed against PAPD7, in order to inhibit expression ofboth, PAPD5 and PAPD7. In this context the shRNA molecule in (a) may beone or more of the following shRNA molecules

(SEQ ID NO: 260) CCGGGCCACATATAGAGATTGGATACTCGAGTATCCAATCTCTATATGTGGCTTTTTG (SEQ ID NO: 261)CCGGCCAACAAATCTCAGCATGGATCTCGAGATCCA TGCTGAGATTTGTTGGTTTTTG(SEQ ID NO: 262) CCGGCGCCTGTAATCCCAGCACTTTCTCGAGAAAGTGCTGGGATTACAGGCGTTTTTG (SEQ ID NO: 263)CCGGGCCTGTAATCCCAGCACTTTACTCGAGTAAAG TGCTGGGATTACAGGCTTTTTG(SEQ ID NO: 264) CCGGCGATGTTGGAAGGAGTTCATACTCGAGTATGAACTCCTTCCAACATCGTTTTTG

In a further aspect the invention the RNAi molecule is an antisenseoligonucleotide capable of inhibiting expression of PAPD5 or PAPD7. Themodulation is achieved by hybridizing to a target nucleic acid encodingPAPD5 or PAPD7. The target nucleic acid may be a mammalian PAPD5, suchas a sequence selected from the group consisting of SEQ ID NO: 4, 5 and10, or natural variants thereof.

The target nucleic acid may be a mammalian PAPD7, such as a sequenceselected from SEQ ID NO: 6 or 11 or natural variants thereof.

The oligonucleotide of the invention is an antisense oligonucleotidewhich targets PAPD5 or PAPD7.

In some embodiments the antisense oligonucleotide of the invention iscapable of modulating the expression of the target by inhibiting ordown-regulating it. Preferably, such modulation produces an inhibitionof expression of at least 20% compared to the normal expression level ofthe target, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, or90% inhibition compared to the normal expression level of the target. Insome embodiments oligonucleotides of the invention may be capable ofinhibiting expression levels of PAPD5 or PAPD7 mRNA by at least 60% or70% in vitro using HeLa cells or HepaRG cells. In some embodimentscompounds of the invention may be capable of inhibiting expressionlevels of PAPD5 or PAPD7 protein by at least 50% in vitro using HeLacells or HepaRG cells. Suitable, assays which may be used to measurePAPD5 or PAPD7 RNA or protein inhibition are described under thescreening methods above. The target modulation is triggered by thehybridization between a contiguous nucleotide sequence of theoligonucleotide and the target nucleic acid.

An aspect of the present invention relates to an antisenseoligonucleotide which comprises a contiguous nucleotide sequence of 10to 30 nucleotides in length wherein the contiguous nucleotide sequenceis at least 90% complementarity to PAPD5. The antisense oligonucleotideis capable of reducing expression of PAPD5. Preferably the antisenseoligonucleotide comprise at least one 2′ sugar modified nucleoside.

Another aspect of the present invention relates to an antisenseoligonucleotide which comprises a contiguous nucleotide sequence of 10to 30 nucleotides in length wherein the contiguous nucleotide sequenceis at least 90% complementarity to PAPD7. The antisense oligonucleotideis capable of reducing expression of PAPD7. Preferably the antisenseoligonucleotide comprise at least one 2′ sugar modified nucleoside.

In some embodiments, the oligonucleotide comprises a contiguous sequencewhich is at least 90% complementary, such as at least 91%, such as atleast 92%, such as at least 93%, such as at least 94%, such as at least95%, such as at least 96%, such as at least 97%, such as at least 98%,or 100% complementary with a region of the target nucleic acid or atarget sequence.

In a preferred embodiment the oligonucleotide of the invention, orcontiguous nucleotide sequence thereof is fully complementary (100%complementary) to a region of the target nucleic acid, or in someembodiments may comprise one or two mismatches between theoligonucleotide and the target nucleic acid.

In some embodiments the oligonucleotide comprises a contiguousnucleotide sequence of 10 to 30 nucleotides in length with at least 90%complementary, such as fully (or 100%) complementary, to a targetnucleic acid region, such as a target sequence, present in SEQ ID NO: 4,5 or 10. In some embodiments the oligonucleotide sequence is 100%complementary to a corresponding target nucleic acid region present inSEQ ID NO: 10. In some embodiments the oligonucleotide sequence is 100%complementary to a corresponding target nucleic acid region present SEQID NO: 4, 5 or 10.

In some embodiments the oligonucleotide comprises a contiguousnucleotide sequence of 10 to 30 nucleotides in length with at least 90%complementary, such as fully (or 100%) complementary, to a targetnucleic acid region, such as a target sequence, present in SEQ ID NO: 6or 11. In some embodiments the oligonucleotide sequence is 100%complementary to a corresponding target nucleic acid region present inSEQ ID NO: 11. In some embodiments the oligonucleotide sequence is 100%complementary to a corresponding target nucleic acid region present SEQID NO: 6 or 11.

In some embodiments, the oligonucleotide of the invention comprises orconsists of 10 to 35 nucleotides in length, such as from 10 to 30, suchas 11 to 22, such as from 12 to 20, such as from such as from 14 to 20,such as from 14 to 18 such as from 14 to 16, such as from 16 to 20contiguous nucleotides in length.

In some embodiments, the oligonucleotide or contiguous nucleotidesequence thereof comprises or consists of 22 or less nucleotides, suchas 20 or less nucleotides. It is to be understood that any range givenherein includes the range endpoints. Accordingly, if an oligonucleotideis said to include from 10 to 30 nucleotides, both 10 and 30 nucleotidesare included.

In some embodiments, the oligonucleotide or contiguous nucleotidesequence comprises or consists of a sequence selected from the groupconsisting of sequences listed in table 3, in the materials and methodsection.

In some embodiments, the antisense oligonucleotide or contiguousnucleotide sequence comprises or consists of 10 to 30 nucleotides inlength with at least 90% identity, preferably 100% identity, to asequence selected from the group consisting of SEQ ID NO: 12 to 131 (seemotif sequences listed in table 3).

In some embodiments, the antisense oligonucleotide or contiguousnucleotide sequence comprises or consists of 10 to 30 nucleotides inlength with at least 90% identity, preferably 100% identity, to asequence selected from the group consisting of SEQ ID NO: 15, 18, 23,25, 26, 30, 32, 39, 54, 56, 58, 65, 80, 88, 92, 93, 111, 115, 116 and118 (see motif sequences listed in table 3).

In some embodiments, the antisense oligonucleotide or contiguousnucleotide sequence comprises or consists of 10 to 30 nucleotides inlength with at least 90% identity, preferably 100% identity, to asequence selected from the group consisting of SEQ ID NO: 23, 26, 54,56, 80, 93 and 115.

In some embodiments, the oligonucleotide or contiguous nucleotidesequence comprises or consists of a sequence selected from the groupconsisting of sequences listed in table 4, in the materials and methodsection.

In some embodiments, the antisense oligonucleotide or contiguousnucleotide sequence comprises or consists of 10 to 30 nucleotides inlength with at least 90% identity, preferably 100% identity, to asequence selected from the group consisting of SEQ ID NO: 132 to 251(see motif sequences listed in table 4).

In some embodiments, the antisense oligonucleotide or contiguousnucleotide sequence comprises or consists of 10 to 30 nucleotides inlength with at least 90% identity, preferably 100% identity, to asequence selected from the group consisting of SEQ ID NO: 153, 155, 168,171, 172, 174, 183, 184, 188, 190, 191, 194, 195, 197, 221, 224, 229,232, 239, and 244 (see motif sequences listed in table 4).

In some embodiments, the antisense oligonucleotide or contiguousnucleotide sequence comprises or consists of 10 to 30 nucleotides inlength with at least 90% identity, preferably 100% identity, to asequence selected from the group consisting of SEQ ID NO: 172, 188, 190,229 and 239.

In a further aspect the invention relates to a combined preparationcomprising a) a nucleic acid molecule which inhibits expression and/oractivity of PAPD5; and b) a nucleic acid molecule which inhibitsexpression and/or activity of PAPD7. In particular embodiments thenucleic acid molecules are independently selected from siRNA, shRNA andantisense oligonucleotides described herein.

In some embodiments the combined preparation comprises a) one of moresiRNA molecules targeting a PAPD5 target sequence selected from one ormore of SEQ ID NO: 252, 253, 254 and 255; and b) one of more siRNAmolecules targeting a PAPD7 target sequence selected from one or more ofSEQ ID NO: 256, 257, 258 and 259.

In some embodiments the combined preparation comprises a) one of moresiRNA molecules targeting a PAPD5 target sequence selected from one ormore of SEQ ID NO: 252, 253, 254 and 255; and b) and b) one of moreantisense oligonucleotides targeting a PAPD7 target sequence selectedfrom the group consisting of SEQ ID NO: 153, 155, 168, 171, 172, 174,183, 184, 188, 190, 191, 194, 195, 197, 221, 224, 229, 232, 239, and244.

In some embodiments the combined preparation comprises a) one of moreantisense oligonucleotide molecules targeting a PAPD5 target sequenceselected from the group consisting of SEQ ID NO: 15, 18, 23, 25, 26, 30,32, 39, 54, 56, 58, 65, 80, 88, 92, 93, 111, 115, 116 and 118; and b)one of more siRNA molecules targeting a PAPD7 target sequence selectedfrom one or more of SEQ ID NO: 256, 257, 258 and 259.

In some embodiments the combined preparation comprises a) one of moreshRNA molecules targeting a PAPD5 target sequence selected from one ormore of SEQ ID NO: 260, 261, 262, 263 and 264; and b) one of more siRNAmolecules targeting a PAPD7 target sequence selected from one or more ofSEQ ID NO: 256, 257, 258 and 259.

In some embodiments the combined preparation comprises a) one of moreshRNA molecules targeting a PAPD5 target sequence selected from one ormore of SEQ ID NO: 260, 261, 262, 263 and 264; and b) one of moreantisense oligonucleotides targeting a PAPD7 target sequence selectedfrom the group consisting of SEQ ID NO: 153, 155, 168, 171, 172, 174,183, 184, 188, 190, 191, 194, 195, 197, 221, 224, 229, 232, 239, and244.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence selected from the group consisting of SEQID NO: 15, 18, 23, 25, 26, 30, 32, 39, 54, 56, 58, 65, 80, 88, 92, 93,111, 115, 116 and 118 and b) an antisense oligonucleotide or contiguousnucleotide sequence comprises or consists of 10 to 30 nucleotides inlength with at least 90% identity, preferably 100% identity, to asequence selected from the group consisting of SEQ ID NO: 153, 155, 168,171, 172, 174, 183, 184, 188, 190, 191, 194, 195, 197, 221, 224, 229,232, 239, and 244.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence selected from the group consisting of SEQID NO: 18, 23, 25, 26, 32, 39, 54, 56, 80, 92, 93, 116 and 118 and b) anantisense oligonucleotide or contiguous nucleotide sequence comprises orconsists of 10 to 30 nucleotides in length with at least 90% identity,preferably 100% identity, to a sequence selected from the groupconsisting of SEQ ID NO: 153, 155, 172, 174, 183, 188, 190, 195, 197,221, 224, 229, 232 and 244.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 18 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 221.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 23 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 172 or 188.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 25 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 174 or 183.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 26 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 183.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 39 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 229.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 54 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 190 or 232.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 56 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 153 or 244.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 80 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 153 or 244.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 92 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 190 or 232.

In some embodiments the combined preparation comprises a) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence SEQ ID NO: 116 and b) an antisenseoligonucleotide or contiguous nucleotide sequence comprises or consistsof 10 to 30 nucleotides in length with at least 90% identity, preferably100% identity, to a sequence of SEQ ID NO: 155 or 195.

Oligonucleotide Design

Oligonucleotide design refers to the pattern of nucleoside sugarmodifications in the oligonucleotide sequence. The oligonucleotides ofthe invention comprise sugar-modified nucleosides and may also compriseDNA or RNA nucleosides. In some embodiments, the oligonucleotidecomprises sugar-modified nucleosides and DNA nucleosides. Incorporationof modified nucleosides into the oligonucleotide of the invention mayenhance the affinity of the oligonucleotide for the target nucleic acid.In that case, the modified nucleosides can be referred to as affinityenhancing modified nucleotides. The modified nucleosides may also betermed units.

In an embodiment, the oligonucleotide comprises at least 1 modifiednucleoside, such as from 1 to 8 modified nucleosides, such as from 2 to8 modified nucleosides, such as from 3 to 7 modified nucleosides, suchas from 4 to 6 modified nucleosides.

In an embodiment, the oligonucleotide comprises one or more sugarmodified nucleosides, such as 2′ sugar modified nucleosides. Preferablythe oligonucleotide of the invention comprise the one or more 2′ sugarmodified nucleoside independently selected from the group consisting of2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA,2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANAand LNA nucleosides. Even more preferably the one or more modifiednucleoside is a locked nucleic acid (LNA).

In a further embodiment the oligonucleotide comprises at least onemodified internucleoside linkage. In a preferred embodiment all theinternucleoside linkages within the contiguous nucleotide sequence arephosphorothioate or boranophosphate internucleoside linkages. In someembodiments all the internucleotide linkages in the contiguous sequenceof the oligonucleotide are phosphorothioate linkages.

In some embodiments, the oligonucleotide of the invention comprises atleast one LNA nucleoside, such as from 1 to 8 LNA nucleosides, such asfrom 2 to 8 LNA nucleosides, such as from 3 to 7 LNA nucleosides, suchas from 4 to 6 LNA nucleosides.

In some embodiments, the oligonucleotide of the invention comprises atleast one LNA nucleoside and at least one 2′ substituted modifiednucleoside.

In an embodiment of the invention the oligonucleotide of the inventionis capable of recruiting RNase H.

Gapmer Design

In a preferred embodiment the oligonucleotide of the invention has agapmer design or structure also referred herein merely as “Gapmer”. In agapmer structure the oligonucleotide comprises at least three distinctstructural regions a 5′-flank, a gap and a 3′-flank, F-G-F′ in '5→3′orientation. In this design, flanking regions F and F′ (also termed wingregions) comprise a contiguous stretch of modified nucleosides, whichare complementary to the PAPD5 or PAPD7 target nucleic acid, while thegap region, G, comprises a contiguous stretch of nucleotides which arecapable of recruiting a nuclease, preferably an endonuclease such asRNase, for example RNase H, when the oligonucleotide is in duplex withthe target nucleic acid. In preferred embodiments the gap regionconsists of DNA nucleosides. Regions F and F′, flanking the 5′ and 3′ends of region G, preferably comprise non-nuclease recruitingnucleosides (nucleosides with a 3′ endo structure), more preferably oneor more affinity enhancing modified nucleosides. In some embodiments,the 3′ flank comprises at least one LNA nucleoside, preferably at least2 LNA nucleosides. In some embodiments, the 5′ flank comprises at leastone LNA nucleoside. In some embodiments both the 5′ and 3′ flankingregions comprise a LNA nucleoside. In some embodiments all thenucleosides in the flanking regions are LNA nucleosides. In otherembodiments, the flanking regions may comprise both LNA nucleosides andother nucleosides (mixed flanks), such as DNA nucleosides and/or non-LNAmodified nucleosides, such as 2′ substituted nucleosides. In this casethe gap is defined as a contiguous sequence of at least 5 RNase Hrecruiting nucleosides (nucleosides with a 2′ endo structure, preferablyDNA) flanked at the 5′ and 3′ end by an affinity enhancing modifiednucleoside, preferably LNA, such as beta-D-oxy-LNA. Consequently, thenucleosides of the 5′ flanking region and the 3′ flanking region whichare adjacent to the gap region are modified nucleosides, preferablynon-nuclease recruiting nucleosides or high affinity nucleosides.

Region F

Region F (5′ flank or 5′ wing) is attached to the 5′ end of region G andcomprises, contains or consists of at least one modified nucleoside suchas at least 2, at least 3, or at least 4 modified nucleosides. In anembodiment region F comprises or consists of from 1 to 4 modifiednucleosides, such as from 2 to 4 modified nucleosides, such as from 1 to3 modified nucleosides, such as 1, 2, 3 or 4 modified nucleosides. The Fregion is defined by having at least on modified nucleoside at the 5′end and at the 3′ end of the region.

In some embodiments, the modified nucleosides in region F have a 3′ endostructure.

In an embodiment, one or more of the modified nucleosides in region Fare 2′ modified nucleosides. In one embodiment all the nucleosides inRegion F are 2′ modified nucleosides.

In another embodiment region F comprises DNA and/or RNA nucleosides inaddition to the 2′ modified nucleosides. Flanks comprising DNA and/orRNA are characterized by having a 2′ modified nucleoside in the 5′ endand the 3′end (adjacent to the G region) of the F region. The DNAnucleosides in the flanks should preferably not be able to recruit RNaseH. The length of the 5′ flank (region F) in oligonucleotides with DNAand/or RNA nucleotides in the flanks may be longer, maintaining thenumber of 2′ modified nucleotides at 1 to 4 as described above. In afurther embodiment one or more of the 2′ modified nucleosides in regionF are selected from 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNAunits, 2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, LNA units, arabinonucleic acid (ANA) units and 2′-fluoro-ANA units.

In some embodiments the F region comprises both LNA and a 2′ substitutedmodified nucleoside. These are often termed mixed wing or mixed flankoligonucleotides.

In one embodiment of the invention all the modified nucleosides inregion F are LNA nucleosides. In a further embodiment all thenucleosides in region F are LNA nucleosides. In a further embodiment theLNA nucleosides in region F are independently selected from the groupconsisting of oxy-LNA, thio-LNA, amino-LNA, cET, and/or ENA, in eitherthe beta-D or alpha-L configurations or combinations thereof. In apreferred embodiment region F comprises at least 1 beta-D-oxy LNA unit,at the 5′ end of the contiguous sequence. In a further preferredembodiment region F consists of beta-D-oxy LNA nucleosides.

Region G

Region G (gap region) preferably comprise, contain or consist of from 4to 18, or from 5 to 17, or from 6 to 16 or from 8 to 12 consecutivenucleotide units capable of recruiting RNase H nuclease.

The nucleoside units in region G, which are capable of recruitingnuclease are in an embodiment selected from the group consisting of DNA,alpha-L-LNA, C4′ alkylated DNA (as described in PCT/EP2009/050349 andVester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, bothincorporated herein by reference), arabinose derived nucleosides likeANA and 2′F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661), UNA(unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst.,2009, 10, 1039 incorporated herein by reference). UNA is unlockednucleic acid, typically where the bond between C2 and C3 of the ribosehas been removed, forming an unlocked “sugar” residue.

In some embodiments, region G consists of 100% DNA units.

In further embodiments the region G may consist of a mixture of DNA andother nucleosides capable of mediating RNase H cleavage.

In some embodiments, nucleosides in region G have a 2′ endo structure.

Region F′

Region F′ (3′ flank or 3′ wing) is attached to the 3′ end of region Gand comprises, contains or consists of at least one modified nucleosidesuch as at least 2, at least 3, or at least 4 modified nucleosides. Inan embodiment region F′ comprises or consists of from 1 to 4 modifiednucleosides, such as from 2 to 4 modified nucleosides, such as from 1 to3 modified nucleosides, such as 1, 2, 3 or 4 modified nucleosides. TheF′ region is defined by having at least on modified nucleoside at the 5′end and at the 3′ end of the region.

In some embodiments, the modified nucleosides in region F′ have a 3′endo structure.

In an embodiment, one or more of the modified nucleosides in region F′are 2′ modified nucleosides. In one embodiment all the nucleosides inRegion F′ are 2′ modified nucleosides.

In another embodiment region F′ comprises DNA and/or RNA nucleosides inaddition to the 2′ modified nucleosides. Flanks comprising DNA and/orRNA are characterized by having a 2′ modified nucleoside in the 5′ endand the 3′end (adjacent to the G region) of the F′ region. The DNAnucleosides in the flanks should preferably not be able to recruit RNaseH. The length of the 3′ flank (region F′) in oligonucleotides with DNAand/or RNA nucleotides in the flanks may be longer, maintaining thenumber of 2′ modified nucleotides at 1 to 4 as described above. In afurther embodiment one or more of the 2′ modified nucleosides in regionF′ are selected from 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNAunits, 2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, LNA units, arabinonucleic acid (ANA) units and 2′-fluoro-ANA units.

In some embodiments the F′ region comprises both LNA and a 2′substituted modified nucleoside. These are often termed mixed wing ormixed flank oligonucleotides.

In one embodiment of the invention all the modified nucleosides inregion F′ are LNA nucleosides. In a further embodiment all thenucleosides in region F′ are LNA nucleosides. In a further embodimentthe LNA nucleosides in region F′ are independently selected from thegroup consisting of oxy-LNA, thio-LNA, amino-LNA, cET, and/or ENA, ineither the beta-D or alpha-L configurations or combinations thereof. Ina preferred embodiment region F′ comprises at least two beta-D-oxy LNAunit, at the 3′ end of the contiguous sequence. In a further preferredembodiment region F′ consists of beta-D-oxy LNA nucleosides.

Region D′ and D″

Region D′ and D″ can be attached to the 5′ end of region F or the 3′ endof region F′, respectively.

Region D′ or D″ may independently comprise 1, 2, 3, 4 or 5 additionalnucleotides, which may be complementary or non-complementary to thetarget nucleic acid. In this respect the oligonucleotide of theinvention, may in some embodiments comprise a contiguous nucleotidesequence capable of modulating the target which is flanked at the 5′and/or 3′ end by additional nucleotides. Such additional nucleotides mayserve as a nuclease susceptible biocleavable linker (see definition oflinkers). In some embodiments the additional 5′ and/or 3′ endnucleotides are linked with phosphodiester linkages, and may be DNA orRNA. In another embodiment, the additional 5′ and/or 3′ end nucleotidesare modified nucleotides which may for example be included to enhancenuclease stability or for ease of synthesis. In one embodiment theoligonucleotide of the invention comprises a region D′ and/or D″ inaddition to the contiguous nucleotide sequence.

The gapmer oligonucleotide of the present invention can be representedby the following formulae:

F-G-F′; in particular F₁₋₇-G₄₋₁₂-F′₁₋₇

D′-F-G-F′, in particular D′₁₋₃-F₁₋₇-G₄₋₁₂-F′₁₋₇

F-G-F′-D″, in particular F₁₋₇-G₄₋₁₂-F′₁₋₇-D″₁₋₃

D′-F-G-F′-D″, in particular D′₁₋₃-F₁₋₇-G₄₋₁₂-F₁₋₇-D″₁₋₃

The preferred number and types of nucleosides in regions F, G and F′, D′and D″ have been described above.

In some embodiments the oligonucleotide is a gapmer consisting of 14-20nucleotides in length, wherein each of regions F and F′ independentlyconsists of 1, 2, 3 or 4 modified nucleoside units and region G consistsof 6-17 nucleoside units, capable of recruiting nuclease when in duplexwith the PAPD5 or PAPD7 target nucleic acid and wherein theoligonucleotide is complementary to the PAPD5 or PAPD7 target nucleicacid.

In all instances the F-G-F′ design may further include region D′ and/orD″, which may have 1, 2 or 3 nucleoside units, such as DNA units.Preferably, the nucleosides in region F and F′ are modified nucleosides,while nucleotides in region G are unmodified nucleosides.

In each design, the preferred modified nucleoside is LNA.

In another embodiment all the internucleoside linkages in the gap in agapmer are phosphorothioate and/or boranophosphate linkages. In anotherembodiment all the internucleoside linkages in the flanks (F and F′region) in a gapmer are phosphorothioate and/or boranophosphatelinkages. In another preferred embodiment all the internucleosidelinkages in the D′ and D″ region in a gapmer are phosphodiesterlinkages.

For specific gapmers as disclosed herein, when the cytosine (C) residuesare annotated as 5-methyl-cytosine, in various embodiments, one or moreof the C′s present in the oligonucleotide may be unmodified C residues.

For certain embodiments of the invention, the oligonucleotide isselected from the group of oligonucleotide compounds with CMP-ID-NO:12_1 to 131_1 (see oligonucleotides listed in table 3).

For certain embodiments of the invention, the oligonucleotide isselected from the group of oligonucleotide compounds with CMP-ID-NO:15_1, 18_1, 23_1, 25_1, 26_1, 30_, 32_1, 39_1, 54_1, 56_1, 58_1, 65_1,80_1, 88_1, 92_1, 93_1, 111_1, 115_1, 116_1 and 118_1 (seeoligonucleotides listed in table 3).

For certain embodiments of the invention, the oligonucleotide isselected from the group of oligonucleotide compounds with CMP-ID-NO:23_1, 26_1, 54_1, 56_1, 80_1, 93_1 and 115_1.

For certain embodiments of the invention, the oligonucleotide isselected from the group of oligonucleotide compounds with CMP-ID-NO: 132to 251_1 (see oligonucleotides listed in table 4).

For certain embodiments of the invention, the oligonucleotide isselected from the group of oligonucleotide compounds with CMP-ID-NO:153_1, 155_1, 168_1, 171_1, 172_1, 174_1, 183_1, 184_1, 188_1, 190_1,191_1, 194_1, 195_1, 197_1, 221_1, 224_1, 229_1, 232_1, 239_1, and 244_1(see oligonucleotides listed in table 4).

For certain embodiments of the invention, the oligonucleotide isselected from the group of oligonucleotide compounds with CMP-ID-NO:172_1, 188_1, 190_1, 229_1 and 237_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide is selected from the group of oligonucleotidecompounds with CMP ID NO: 18_1, 25_1, 26_1, 32_1, 39_1, 54_1, 56_1,80_1, 92_1, 93_1, 116_1 and 118_1 and b) the oligonucleotide is selectedfrom the group of oligonucleotide compounds with CMP ID NO: 153_1,155_1, 168_1, 171_1, 172_1, 174_1, 183_1, 184_1, 188_1, 190_1, 191_1,194_1, 195_1, 197_1, 221_1, 224_1, 229_1, 232_1, 23_19, and 244_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide compound with CMP ID NO: 18_1 and b) theoligonucleotide compound with CMP ID NO: 221_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide compound with CMP ID NO: 23_1 and b) theoligonucleotide compound with CMP ID NO: 172_1 or 188_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide compound with CMP ID NO: 25_1 and b) theoligonucleotide compound with CMP ID NO: 174_1 or 183_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide compound with CMP ID NO: 26_1 and b) theoligonucleotide compound with CMP ID NO: 183_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide compound with CMP ID NO: 39_1 and b) theoligonucleotide compound with CMP ID NO: 229_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide compound with CMP ID NO: 54_1 and b) theoligonucleotide compound with CMP ID NO: 190_1 or 232_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide compound with CMP ID NO: 56_1 and b) theoligonucleotide compound with CMP ID NO: 153_1 or 244_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide compound with CMP ID NO: 80_1 and b) theoligonucleotide compound with CMP ID NO: 153_1 or 244_1.

An embodiment of the invention is a combined preparation comprising a)the oligonucleotide compound with CMP ID NO: 116_1 and b) theoligonucleotide compound with CMP ID NO: 155_1 or 195_1.

Applications

In context of the present invention it has surprisingly been shown thatthe combined inhibition of PAPD5 and PAPD7 leads to a synergistic effectin the inhibition of HBV propagation. The appended examples show thatreduction of the expression of PAPD5 alone leads to a reduction of thesecretion of HBsAg and HBeAg of around 50%, likewise intracellular HBVmRNA was reduced using a PAPD5 inhibitor. Reduction of the expression ofPAPD7 alone leads to a reduction of the secretion of HBsAg and HBeAg ofnot more than 15%. Simultaneous knock-down of PAPD5 and PAPD7 leads to asynergistic effect in the reduction of secretion of HBsAg and HBeAg thatlies above the sum of the single knock-downs. Without being bound bytheory, this synergistic effect may be due to a compensatory effect ofPAPD5 and PAPD7 since both proteins have high sequence homology and sameenzymatic functions. Due to the reduction of HBsAg secretion theinhibitor of the present invention inhibits development of chronic HBVinfection. In particular, due to inhibition of HBeAg secretion, theinhibitor of the present invention more efficiently inhibits developmentof a chronic HBV infection as compared to a compound that only reducessecretion of HBsAg. In addition, reducing HBeAg in an expected mothermay also inhibit the development of a chronic HBV infection of herchild. Thus, due to the reduction of HBeAg secretion the inhibitor ofthe present invention inhibits development of a chronic HBV infection(such as development of a chronic HBV infection in the offspring of anHBV infected mother) and reduces the infectiousness of a HBV infectedperson. Accordingly, one aspect of the present invention related to theherein provided inhibitor, wherein the inhibitor reduces secretion ofHBsAg and HBeAg. In line with this, a further aspect of the inventionrelates to the herein provided inhibitor, in particular a nucleic acidmolecule or a combination of nucleic acid molecules, wherein theinhibitor inhibits development of chronic HBV infection and reduces theinfectiousness of a HBV infected person. In a particular aspect of theinvention, the herein provided inhibitor inhibits development of achronic HBV infection in the offspring of a HBV infected mother. Thismother is preferably HBeAg positive.

The subject to be treated with the inhibitor of the invention (or whichprophylactically receives the inhibitor of the present invention) ispreferably a human, more preferably a human patient who is HBsAgpositive and/or HBeAg positive, even more preferably a human patientthat is HBsAg positive and HBeAg positive. Said human patient may be anexpected mother, e.g. an expected mother who is HBeAg positive and/orHBsAg positive, more preferably an expected mother who is HBeAg positiveand HBsAg positive.

One embodiment of the present invention relates a PAPD5 inhibitor, inparticular a nucleic acid molecule that inhibits the expression and/oractivity of PAPD5, for use in the treatment and/or prevention of a HBVinfection, in particular a chronic HBV infection. A further embodimentof the present invention relates to a combined preparation comprising aninhibitor of PAPD5 and an inhibitor of PAPD7 for use in the treatmentand/or prevention of a HBV infection, in particular a chronic HBVinfection. In a preferred embodiment the combined composition for use intreatment and/or prevention of a HBV infection comprises a) a nucleicacid molecule which inhibits expression and/or activity PAPD5; and b) anucleic acid molecule which inhibits expression and/or activity ofPAPD7. Thus, the present invention relates to a combined preparationcomprising an inhibitor of PAPD5 and an inhibitor of PAPD7 forsimultaneous or sequential use in the treatment and/or prevention of aHBV infection.

The present invention also relates to a combined preparation comprisinga) a nucleic acid molecule which inhibits expression and/or activityPAPD5; and b) a nucleic acid molecule which inhibits expression and/oractivity of PAPD7. It is envisaged in context of the invention that saidcombined preparation is used for treating (e.g. ameliorating) a HBVinfection. The definitions disclosed herein in connection with theinhibitor of the present invention apply, mutatis mutandis, to thecombined preparation of the present invention. The combined preparationmay comprise a molecule that is a PAPD5 inhibitor and a separatemolecule that is a PAPD7 inhibitor (e.g. two separate RNAi molecules,such as siRNA molecules, shRNA and antisense oligonucleotides, or twoseparate small molecules). These two separate inhibitors may beformulated within one unit, e.g., within one pill or vial.Alternatively, these two separate inhibitors may be formulatedseparately, in separate units, e.g. separate pills or vials. The twoseparate inhibitors may be administered together, (i.e. simultaneously)or separately (i.e. sequentially) provided that the synergistic effectof the two inhibitors is achieved. In one aspect of the invention thecombined preparation leads to a reduction of secretion of HBsAg andHBeAg of at least 50% as compared to the no drug control (i.e. comparedto cells or subjects to which no drug is administrated).

The present invention also relates to a pharmaceutical composition foruse in the treatment and/or prevention of a HBV infection, wherein thepharmaceutical composition comprises

(i) the inhibitor of the invention; or the combined preparation of theinvention; and

(ii) optionally a pharmaceutically acceptable carrier.

Accordingly, the present invention relates to a method of treatingand/or preventing a HBV infection, wherein the method comprisesadministering an effective amount of the inhibitor of the invention, inparticular a nucleic acid molecule, a conjugate of the inhibitor, thepharmaceutical composition of the invention, or of the combinedpreparation of the invention to a subject in need of such a treatment.

The invention also provides for the use of the inhibitor of theinvention, in particular a nucleic acid molecule, a conjugate of theinhibitor, the pharmaceutical composition of the invention, or of thecombined preparation of the invention for the manufacture of amedicament. In preferred embodiments the medicament is manufactured in adosage form for subcutaneous administration and for the combinedpreparation the ratio of the PAPD5 inhibitor and the PAPD7 inhibitor is1:1 by weight.

The invention also provides for the use of the inhibitor of theinvention, in particular a nucleic acid molecule, a conjugate of theinhibitor, the pharmaceutical composition of the invention, or of thecombined preparation of the invention as described for the manufactureof a medicament wherein the medicament is in a dosage form forintravenous administration and for the combined preparation the ratio ofthe PAPD5 inhibitor and the PAPD7 inhibitor is 1:1 by weight.

The inhibitor of the invention, the combined preparation of theinvention, or the pharmaceutical composition of the invention may beused in a combination therapy. For example, the inhibitor of theinvention, the combined preparation of the invention, or thepharmaceutical composition of the invention may be combined with otheranti-HBV agents such as interferon alpha-2b, interferon alpha-2a, andinterferon alphacon-1 (pegylated and unpegylated), ribavirin, lamivudine(3TC), entecavir, tenofovir, telbivudine (LdT), adefovir, or otheremerging anti-HBV agents such as a HBV RNA replication inhibitor, aHBsAg secretion inhibitor, a HBV capsid inhibitor, an antisense oligomer(e.g. as described in WO2012/145697 and WO 2014/179629), a siRNA (e.g.described in WO 2005/014806, WO 2012/024170, WO 2012/2055362, WO2013/003520, WO 2013/159109, WO 2017/027350 and W02017/015175), a HBVtherapeutic vaccine, a HBV prophylactic vaccine, a HBV antibody therapy(monoclonal or polyclonal), or TLR 2, 3, 7, 8 or 9 agonists for thetreatment and/or prophylaxis of HBV.

The appended examples demonstrate that down regulation of PAPD5 and/orPAPD7 goes along with a reduction in the production of HBsAg and HBeAgas well as of intracellular HBV mRNA in HBV infected cells. Theseresults indicate that the amount and/or activity of PAPD5 and/or PAPD7can be used for monitoring therapeutic success during the treatment of aHBV infection, e.g. if treatment with an inhibitor of PAPD5 and/or PAPD7is ongoing or has been performed. Thus, the present invention relates toa method for monitoring the therapeutic success during the treatment ofa HBV infection, wherein the method comprises:

(a) analyzing in a sample obtained from a test subject the amount and/oractivity of PAPD5 and/or PAPD7;

(b) comparing said amount and/or activity with reference datacorresponding to the amount and/or activity of PAPD5 and/or PAPD7 of atleast one reference subject; and

(c) predicting therapeutic success based on the comparison step (b).

In the monitoring method of the invention the test subject may be ahuman being who receives medication for a HBV infection or has receivedmedication for a HBV infection. The medication may comprise anti-HBVagents as described above. The medication may also comprise an inhibitorof PAPD5 and/or PAPD7.

In the monitoring method of the invention the reference data maycorrespond to the amount and/or activity of PAPD5 and/or PAPD7 in asample of at least one reference subject. Said sample may be blood or aliver biopsy.

One aspect of the invention relates to the monitoring method of theinvention, wherein the at least one reference subject has a HBVinfection but did not receive medication for a HBV infection; andwherein in step (c) a decreased amount and/or activity of PAPD5 and/orPAPD7 of the test subject as compared to the reference data indicatestherapeutic success in the treatment of a HBV infection. For example,said decreased amount and/or activity of PAPD5 and/or PAPD7 may meanthat the amount and/or activity of PAPD5 and/or PAPD7 in the sample ofthe test subject is 0 to 90% of the amount and/or activity of PAPD5and/or PAPD7 in the sample of the at least one reference subject. Forexample, said decreased amount and/or activity of PAPD5 and/or PAPD7 maybe 0 to 80%, preferably 0 to 70%, more preferably 0 to 60%, even morepreferably 0 to 50%, even more preferably 0 to 40%, even more preferably0 to 30, even more preferably 0 to 20%, and most preferably 0 to 10% ofthe amount and/or activity of PAPD5 and/or PAPD7 in the sample of the atleast one reference subject.

Another aspect of the invention relates to the monitoring method of theinvention, wherein the at least one reference subject has a HBVinfection and has received medication for a HBV infection; and whereinin step (c) an identical or similar amount and/or activity of PAPD5and/or PAPD7 of the test subject as compared to the reference dataindicates therapeutic success in the treatment of a HBV infection. Afurther aspect of the invention relates to the monitoring method of theinvention, wherein the at least one reference subject does not have aHBV infection; and wherein in step (c) an identical or similar amountand/or activity of PAPD5 and/or PAPD7 of the test subject as compared tothe reference data indicates therapeutic success in the treatment of aHBV infection. An identical or similar amount and/or activity of PAPD5and/or PAPD7 may mean that the amount and/or activity of PAPD5 and/orPAPD7 in the sample of the test subject is 90-110% of the amount and/oractivity of PAPD5 and/or PAPD7 in the sample of the at least onereference subject. For example, said identical or similar amount and/oractivity of PAPD5 and/or PAPD7 may be 95-105% of the amount and/oractivity of PAPD5 and/or PAPD7 in the sample of the at least onereference subject.

Also encompassed by the present invention is a cell or a non-humananimal (e.g. a mouse, rat, ferret or rabbit) with increased, reduced orabsent PAPD5 and/or PAPD7 expression that can be used for identifyingand/or characterizing a compound that prevents and/or treats (e.g.ameliorates) a HBV infection. For example, said cell or non-human animalmay comprise an exogenous nucleotide sequence encoding PAPD5 and/orPAPD7, e.g. cloned into an expression vector and operable linked to anexogenous promoter. Said cell or non-human animal may overexpress PAPD5and/or PAPD7, preferably PAPD5 and PAPD7. Alternatively, said cell ornon-human animal may have a knock-down of PAPD5 and/or PAPD7, preferablyof PAPD5 and PAPD7.

Embodiments of the Invention

Thus, the present invention relates to the following items:

1. A method for identifying a compound that prevents, ameliorates and/orinhibits a hepatitis B virus (HBV) infection, comprising

-   -   a. contacting a test compound with a cell expressing PAPD5        and/or PAPD7    -   b. measuring the expression and/or activity of PAPD5 and/or        PAPD7 in the presence and absence of said test compound; and    -   c. identifying a compound that reduces the expression and/or        activity of PAPD5 or PAPD7 as a compound that prevents,        ameliorates and/or inhibits a HBV infection.

2. The method of item 1, further comprising the step of testing theability of combinations of compounds identified in c) to reduce theexpression and/or activity of PAPD5 and PAPD7.

3. The method of item 1 or 2, wherein PAPD5 is a PAPD5 target nucleicacid.

4. The method of item 3, wherein the PAPD5 target nucleic acid comprisesor consists of

-   -   a. a nucleotide sequence of SEQ ID NO: 4 or 5 or 10, or natural        variant thereof;    -   b. a nucleotide sequence having at least 80% identity to a        nucleotide sequence of a.), wherein the polypeptide expressed        from the nucleic acid sequence has poly-A polymerase function;    -   c. a nucleotide sequence comprising or consisting of SEQ ID NO:        4, 5 or 10;    -   d. a nucleotide sequence encoding the amino acid sequence of SEQ        ID NO: 1 or 2;    -   e. a nucleotide sequence encoding an amino acid sequence having        at least 80% identity to SEQ ID NO: 1 or 2, wherein the        polynucleotide encodes a polypeptide that has poly-A polymerase        function;    -   f. a nucleotide sequence encoding an enzymatically active        fragment of SEQ ID NO: 1 or 2, such as SEQ ID NO: 7 or 8; or    -   g. a nucleotide sequence encoding an amino acid sequence having        at least 80% identity to an amino acid sequence of an        enzymatically active fragment of SEQ ID NO: 1 or 2, such as SEQ        ID NO: 7 or 8, wherein the polynucleotide encodes a polypeptide        that has poly-A polymerase function;

5. The method of item 1 or 2, wherein PAPD7 is a PAPD7 target nucleicacid.

6. The method of item 5, wherein the PAPD7 target nucleic acid comprisesor consists of

-   -   a. a nucleotide sequence of SEQ ID NO: 6 or 11, or natural        variant thereof;    -   b. a nucleotide sequence having at least 80% identity to a        nucleotide sequence of (a.), wherein the polypeptide expressed        from the nucleic acid sequence has poly-A polymerase function;    -   c. a nucleotide sequence comprising or consisting of SEQ ID NO:        6 or 11;    -   d. the nucleotide sequence encoding the amino acid sequence of        SEQ ID NO: 3;    -   e. a nucleotide sequence encoding an amino acid sequence having        at least 80% identity to SEQ ID NO: 3, wherein the        polynucleotide encodes a polypeptide that has poly-A polymerase        function;    -   f. the nucleotide sequence encoding an enzymatically active        fragment of SEQ ID NO: 3, such as SEQ ID NO: 9; or    -   g. a nucleotide sequence encoding an amino acid sequence having        at least 80% identity to an amino acid sequence of an        enzymatically active fragment of SEQ ID NO: 3, such as SEQ ID        NO: 9, wherein the polynucleotide encodes a polypeptide that has        poly-A polymerase function.

7. The method of any one of items 1 to 6, wherein said cell is aneukaryotic cell.

8. The method of any one of items 1 to 7, wherein the compound thatinhibits propagation of HBV inhibits secretion of HBV surface antigen(HBsAg), inhibits secretion of HBV envelope antigen (HBeAg), and/orinhibits production of intracellular HBV mRNA or HBV DNA.

9. The method of any one of items 1 to 8, wherein the test compound is ascreening library of nucleic acid molecules selected from

-   -   a. single stranded antisense oligonucleotides, or    -   b. siRNA molecules; and    -   c. shRNA molecules.

10. The method of any one of items 1 to 9, wherein the compoundidentified in step c. of item 1 reduce PAPD5 or PAPD7 mRNA expression byat least 50%.

11. The method of any one of items 1 to 10, wherein the test compound isa combined preparation of a nucleic acid molecule capable of reducingPAPD5 and a nucleic acid molecule capable of reducing PAPD7.

12. The method of item 11, wherein the combined preparation reduce HBVsurface antigen (HBsAg), HBV envelope antigen (HBeAg), and/orintracellular HBV mRNA or HBV DNA by at least 70%.

13. The method of any one of items 1 to 12, which additionally comprisesthe step of comparing the test compound to a control.

14. The method of item 13, wherein said control is an inactive testcompound that does not reduce the expression and/or activity of PAPD5 orPAPD7.

15. The method of any one of items 1 to 14, wherein the activity ofPAPD5 and PAPD7 is the poly-A polymerase function.

16. An inhibitor of PAPD5 or PAPD7 for use in treating and/or preventinga HBV infection, wherein said inhibitor is

-   -   a. a RNA interference (RNAi) molecule against PAPD5 or PAPD7; or    -   b. a genome editing machinery, comprising:        -   i. a site-specific DNA nuclease or a polynucleotide encoding            a site-specific DNA nuclease; and        -   ii. a guide RNA or a polynucleotide encoding a guide RNA.

17. The inhibitor for the use according to item 16, wherein theinhibitor is an RNAi molecule selected from the group consisting of:

-   -   a. a single stranded antisense oligonucleotide;    -   b. a siRNA molecule; and    -   c. a shRNA molecule.

18. The inhibitor for the use according to item 16 or 17, wherein theinhibitor is a combined preparation comprising

-   -   a. a RNAi molecule which inhibits expression and/or activity of        PAPD5; and    -   b. a RNAi molecule which inhibits expression and/or activity of        PAPD7.

19. The inhibitor for the use according to any one of items 16 to 18,wherein the inhibitor reduces secretion of HBsAg and HBeAg.

20. The inhibitor for the use according to item any one of items 16 to18, wherein the inhibitor reduces production of intracellular HBV mRNAor HBV DNA.

21. The inhibitor for the use according to any one of items 16 to 20,wherein the inhibitor inhibits development of chronic HBV infectionand/or reduces the infectiousness of a HBV infected person.

22. An antisense oligonucleotide or siRNA molecule which comprises orconsists of a contiguous nucleotide sequence of 10 to 30 nucleotides inlength, wherein the contiguous nucleotide sequence is at least 80%complementarity to PAPD5 target nucleic acid and the antisenseoligonucleotide capable of reducing expression of PAPD5.

23. A nucleic acid molecule which comprises or consists of a contiguousnucleotide sequence of 10 to 30 nucleotides in length wherein thecontiguous nucleotide sequence is at least 80% complementarity to PAPD7target nucleic acid and the antisense oligonucleotide capable ofreducing expression of PAPD7.

24. The nucleic acid molecule of item 23, wherein the nucleic acidmolecule is a single stranded antisense oligonucleotide.

25. The antisense oligonucleotide of item 22, wherein theoligonucleotide is capable of hybridizing to a target nucleic acid ofselected from the group consisting of SEQ ID NO: 4, 5 and 10 with a ΔG°below −10 kcal.

26. The antisense oligonucleotide of item 23 or 24, wherein theoligonucleotide is capable of hybridizing to a target nucleic acid ofselected from SEQ ID NO: 6 or 11 with a ΔG° below −10 kcal.

27. The antisense oligonucleotide of any one of items 22 to 26, whereinthe target nucleic acid is RNA.

28. The antisense oligonucleotide of item 27, wherein the RNA is mRNA.

29. The antisense oligonucleotide of item 28, wherein the mRNA ispre-mRNA or mature mRNA.

30. The antisense oligonucleotide of any one of items 22-29, wherein thecontiguous nucleotide sequence comprises or consists of from 12 to 22nucleotides.

31. The antisense oligonucleotide of item 30, wherein the contiguousnucleotide sequence comprises or consists of from 14-20 nucleotides.

32. The antisense oligonucleotide of any one of items 22-31, wherein theantisense oligonucleotide comprises or consists of 12 to 25 nucleotidesin length.

33. The antisense oligonucleotide of any one of items 22-32, wherein theoligonucleotide or contiguous nucleotide sequence is single stranded.

34. The antisense oligonucleotide of any one of items 22-33 wherein theoligonucleotide is neither siRNA nor self-complementary.

35. The antisense oligonucleotide of any one of items 22 or 25, whereinthe contiguous nucleotide sequence comprises or consists of a sequenceselected from SEQ ID NO: 12-131.

36. The antisense oligonucleotide of item 35, wherein the contiguousnucleotide sequence comprises or consists of a sequence selected fromSEQ ID NO: 15, 18, 23, 25, 26, 30, 32, 39, 54, 56, 58, 65, 80, 88, 92,93, 111, 115, 116 and 118.

37. The nucleic acid molecule or antisense oligonucleotide of any one ofitem 23, 24 or 26, wherein the contiguous nucleotide sequence comprisesor consists of a sequence selected from SEQ ID NO: 132-151.

38. The antisense oligonucleotide of item 37, wherein the contiguousnucleotide sequence comprises or consists of a sequence selected fromSEQ ID NO: 153, 155, 168, 171, 172, 174, 183, 184, 188, 190, 191, 194,195, 197, 221, 224, 229, 232, 239, and 244.

39. The antisense oligonucleotide molecule of any one of items 22-38,wherein the contiguous nucleotide sequence has zero to three mismatchescompared to the target nucleic acid it is complementary to.

40. The antisense oligonucleotide of item 39, wherein the contiguousnucleotide sequence has one mismatch compared to the target nucleicacid.

41. The antisense oligonucleotide of item 39, wherein the contiguousnucleotide sequence is fully complementary to the target nucleic acidsequence.

42. The antisense oligonucleotide of any one of items 22-41, comprisingone or more modified nucleosides.

43. The antisense oligonucleotide of item 42, wherein the one or moremodified nucleoside is a high-affinity modified nucleoside.

44. The antisense oligonucleotide of any one of items 22-43, wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.

45. The antisense oligonucleotide of item 44, wherein the modifiedinternucleoside linkage is nuclease resistant.

46. The antisense oligonucleotide of item 44 or 45, wherein at least 50%of the internucleoside linkages within the contiguous nucleotidesequence are phosphorothioate internucleoside linkages orboranophosphate internucleoside linkages.

47. The antisense oligonucleotide of item 44 or 45, wherein all theinternucleoside linkages within the contiguous nucleotide sequence arephosphorothioate internucleoside linkages.

48. The antisense oligonucleotide of any one of items 22-47, wherein theoligonucleotide is capable of recruiting RNase H.

49. The antisense oligonucleotide of item 48, wherein theoligonucleotide is a gapmer.

50. The antisense oligonucleotide of item 48 or 49, wherein theoligonucleotide is a gapmer of formula 5′-F-G-F′-3′, where region F andF′ independently comprise or consist of 1-4 modified nucleosides and Gis a region between 6 and 17 nucleosides which are capable of recruitingRNaseH.

51. The antisense oligonucleotide of any one of items 42-44 or 50,wherein the modified nucleoside is a 2′ sugar modified nucleosideindependently selected from the group consisting of 2′-O-alkyl-IRNA,2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA,2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNAnucleosides.

52. The antisense oligonucleotide of item 50 or 51, wherein one or moreof the modified nucleosides in region F and F′ is a LNA nucleoside.

53. The antisense oligonucleotide of item 52, wherein all the modifiednucleosides in region F and F′ are LNA nucleosides.

54. The antisense oligonucleotide of item 53, wherein region F and F′consist of LNA nucleosides.

55. The antisense oligonucleotide of any one of items 51-54, wherein theLNA nucleoside is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA,beta-D-amino-LNA, alpha-L-amino-LNA, beta-D-thio-LNA, alpha-L-thio-LNA,(S)cET, (R)cET beta-D-ENA and alpha-L-ENA.

56. The oligonucleotide of any one of items 51-54, wherein the LNAnucleoside is oxy-LNA.

57. The antisense oligonucleotide of any one of items 51-56, wherein theLNA nucleoside is beta-D-oxy-LNA.

58. The antisense oligonucleotide of any one of items 51-54, wherein theLNA nucleoside is thio-LNA.

59. The antisense oligonucleotide of any one of items 51-54, wherein theLNA nucleoside is amino-LNA.

60. The antisense oligonucleotide of any one of items 51-54, wherein theLNA nucleoside is cET.

61. The antisense oligonucleotide of any one of items 51-54, wherein theLNA nucleoside is ENA.

62. The antisense oligonucleotide of item 52, wherein at least one ofregion F or F′ further comprises at least one 2′ substituted modifiednucleoside independently selected from the group consisting of2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA,2′-amino-DNA and 2′-fluoro-DNA.

63. The antisense oligonucleotide of any one of items 52-62, wherein theRNaseH recruiting nucleosides in region G are independently selectedfrom DNA, alpha-L-LNA, C4′ alkylated DNA, ANA and 2′ F-ANA and UNA.

64. The antisense oligonucleotide of item 50 or 63, wherein thenucleosides in region G are DNA nucleosides.

65. The antisense oligonucleotide of any one of items 22 or 25, whereinthe antisense oligonucleotide is selected from CMP ID NO: 12_1 to 131_1.

66. The antisense oligonucleotide of item 65, wherein the antisensecompound are selected from CMP ID NO: 15_1, 18_1, 23_1, 25_1, 26_1, 30_,32_1, 39_1, 54_1, 56_1, 58_1, 65_1, 80_1, 88_1, 92_1, 93_1, 111_1,115_1, 116_1 and 118_1.

67. The antisense oligonucleotide of items 24 wherein the antisenseoligonucleotide is selected from CMP ID NO: 132_ to 151_1.

68. The antisense oligonucleotide of item 67, wherein the antisensecompound are selected from CMP ID NO: 153_1, 155_1, 168_1, 171_1, 172_1,174_1, 183_1, 184_1, 188_1, 190_1, 191_1, 194_1, 195_1, 197_1, 221_1,224_1, 229_1, 232_1, 239_1, and 244_1.

69. The siRNA molecule of item 22, wherein the siRNA molecule istargeting a PAPD5 target sequence selected from one or more of SEQ IDNO: 252, 253, 254 and 255

70. The nucleic acid molecule of item 23, wherein the nucleic acidmolecule is an siRNA molecule targeting a PAPD7 target sequence selectedfrom one or more of SEQ ID NO: 256, 257, 258 and 259.

71. A conjugate comprising the antisense oligonucleotide or siRNAaccording to any one of claims 22-70, and at least one conjugate moietycovalently attached to said oligonucleotide.

72. The conjugate of item 71, wherein the conjugate moiety is selectedfrom carbohydrates, cell surface receptor ligands, drug substances,hormones, lipophilic substances, polymers, proteins, peptides, toxins,vitamins, viral proteins or combinations thereof.

73. The conjugate of item 71 or 72, wherein the conjugate moiety iscapable of binding to the asialoglycoprotein receptor.

74. The conjugate of any one of item 71-73, comprising a linker which ispositioned between the antisense oligonucleotide and the conjugatemoiety.

75. The conjugate of item 74, wherein the linker is a physiologicallylabile linker.

76. The conjugate of item 75, wherein the physiologically labile linkeris nuclease susceptible linker.

77. The conjugate of item 75 or 76, wherein the oligonucleotide has theformula D′-F-G-F′ or F-G-F′-D″, wherein F, F′ and G are as defined inany one of items 52-65 and D′ or D″ comprises 1, 2 or 3 DNA nucleosideswith phosphorothioate internucleoside linkages.

78. A combined preparation comprising:

-   -   a. a RNAi molecule which inhibits expression and/or activity of        PAPD5; and    -   b. a RNAi molecule which inhibits expression and/or activity of        PAPD7.

79. The combined preparation of item 78, wherein the RNAi molecules areselected from items 22-70 or a conjugate of any one of items 71-77.

80. The combined preparation of item 78, wherein the RNAi molecule in a)is an antisense compounds of item 36 or 66 and where the RNAi moleculein b) is an antisense compounds of item 38 or 68.

81. A pharmaceutical composition comprising the antisenseoligonucleotide or siRNA molecule of any one of items 22-70, or aconjugate of any one of items 71-77, or a combined preparation of item78-80 and optionally a pharmaceutically acceptable diluent, carrier,salt and/or adjuvant.

82. An in vivo or in vitro method for modulating PAPD5 and/or PAPD7expression in a target cell which is expressing PAPD5 and/or PAPD7, saidmethod comprising administering an antisense oligonucleotide or siRNAmolecule of item 22-70 or a conjugate of item 71-77, or a combinedpreparation of item 78 or 79, or the pharmaceutical composition of item81 in an effective amount to said cell.

83. A method for treating or preventing a disease comprisingadministering a therapeutically or prophylactically effective amount ofan antisense oligonucleotide or siRNA molecule of item 22-70 or aconjugate of item 71-77 or a combined preparation of item 78-80, or thepharmaceutical composition of item 81 to a subject suffering from orsusceptible to the disease.

84. The antisense oligonucleotide or siRNA molecule of item 22-70 or aconjugate of item 71-77 or a combined preparation of item 78-80, or thepharmaceutical composition of item 81, for use as a medicament fortreatment or prevention of a disease in a subject.

85. Use of the oligonucleotide of oligonucleotide or siRNA molecule ofitem 22-70 or a conjugate of item 71-77 or a combined preparation ofitem 78-80, for the preparation of a medicament for treatment orprevention of a disease in a subject.

86. The method, the antisense oligonucleotide or the use of any one ofitems 83-85, wherein the PAPD5 and/or PAPD7 is reduced by at least 30%,or at least or at least 40%, or at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 90%, or at least 95% compared tothe expression without the antisense oligonucleotide or siRNA moleculeof item 22-70 or a conjugate of item 71-77, or a combined preparation ofitem 78-80.

87. The method, the antisense oligonucleotide or the use of items 83-85,wherein the disease is selected from HBV infection, in particularchronic HBV infection.

88. A method for monitoring the therapeutic success during the treatmentof a HBV infection, wherein the method comprises:

-   -   a. analyzing in a sample obtained from a test subject the amount        and/or activity of PAPD5 and/or PAPD7;    -   b. comparing said amount and/or activity with reference data        corresponding to the amount and/or activity of PAPD5 and/or        PAPD7 of at least one reference subject; and    -   c. predicting therapeutic success based on the comparison step        (b).

89. The monitoring method of item 88, wherein the test subject is ahuman being who receives medication for a HBV infection or has receivedmedication for a HBV infection.

90. The monitoring method of item 88 or 89, wherein the reference datacorresponds to the amount and/or activity of PAPD5 and/or PAPD7 in asample of at least one reference subject.

91. The monitoring method of any one of items 88 to 90, wherein the atleast one reference subject has a HBV infection but did not receivemedication for a HBV infection; and wherein in step (c) a decreasedamount and/or activity of PAPD5 and/or PAPD7 of the test subject ascompared to the reference data indicates therapeutic success in thetreatment of a HBV infection.

92. The monitoring method of item 91, wherein said decreased amountand/or activity of PAPD5 and/or PAPD7 means that the amount and/oractivity of PAPD5 and/or PAPD7 in the sample of the test subject is 0 to90% of the amount and/or activity of PAPD5 and/or PAPD7 in the sample ofthe at least one reference subject.

93. The monitoring method of any one of items 88 to 90, wherein the atleast one reference subject has a HBV infection and has receivedmedication for a HBV infection; and wherein in step (c) an identical orsimilar amount and/or activity of PAPD5 and/or PAPD7 of the test subjectas compared to the reference data indicates therapeutic success in thetreatment of a HBV infection.

94. The monitoring method of any one of items 88 to 90, wherein the atleast one reference subject does not have a HBV infection; and whereinin step (c) an identical or similar amount and/or activity of PAPD5and/or PAPD7 of the test subject as compared to the reference dataindicates therapeutic success in the treatment of a HBV infection.

95. The monitoring method of item 93 or 94, wherein said identical orsimilar amount and/or activity of PAPD5 and/or PAPD7 means that theamount and/or activity of PAPD5 and/or PAPD7 in the sample of the testsubject is 90-110% of the amount and/or activity of PAPD5 and/or PAPD7in the sample of the at least one reference subject.

Pharmaceutical Compositions

As described above, the invention relates to a composition comprising aninhibitor of PAPD5 alone or in combination with a PAPD7 inhibitor foruse in treating and/or preventing a HBV infection. The inhibitor ispreferably a nucleic acid molecule as defined herein. Specifically, acombined preparation comprising an inhibitor of PAPD5 and an inhibitorof PAPD7 for use in the treatment and/or prevention of a HBV infectionis contemplated; and a pharmaceutical composition comprising saidinhibitor composition or said combined preparation. Said pharmaceuticalcomposition (i.e. medicament) optionally comprises a pharmaceuticallyacceptable carrier. Said pharmaceutical composition may further comprisea therapeutically acceptable diluent, salt, excipient and/or adjuvant.

A typical pharmaceutical composition is prepared by mixing a PAPD5inhibitor alone or with a PAPD7 inhibitor and a carrier or excipient.Suitable carriers and excipients are well known to those skilled in theart and are described in detail in, e.g., Ansel, Ansel's PharmaceuticalDosage Forms and Drug Delivery Systems, Philadelphia: Lippincott,Williams & Wilkins, 2004; Gennaro, Remington: The Science and Practiceof Pharmacy, Philadelphia: Lippincott, Williams & Wilkins, 2000; andRowe, Handbook of Pharmaceutical Excipients, Chicago, PharmaceuticalPress, 2005. The formulations may also include one or more buffers,stabilizing agents, surfactants, wetting agents, lubricating agents,emulsifiers, suspending agents, preservatives, antioxidants, opaquingagents, glidants, processing aids, colorants, sweeteners, perfumingagents, flavoring agents, diluents and other known additives to improveappearance of the drug or aid in the manufacturing of the pharmaceuticalproduct (i.e., medicament). For example, the pharmaceutical compositionof the invention may be formulated by mixing an inhibitor of PAPD5and/or an inhibitor of PAPD7 at ambient temperature at an appropriatepH, and with the desired degree of purity, with physiologicallyacceptable carriers, i.e., carriers that are non-toxic to recipients atthe dosages and concentrations employed into a suitable administrationform. The pharmaceutical composition of the invention may be sterile.

For nucleic acid molecules suitable formulations are found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed., 1985. For a brief review of methods fordrug delivery, see, e.g., Langer (Science 249:1527-1533, 1990). WO2007/031091 provides further suitable and preferred examples ofpharmaceutically acceptable diluents, carriers and adjuvants (herebyincorporated by reference). Suitable dosages, formulations,administration routes, compositions, dosage forms, combinations withother therapeutic agents, pro-drug formulations are also provided inWO2007/031091.

The compounds according to the present invention may exist in the formof their pharmaceutically acceptable salts. The term “pharmaceuticallyacceptable salt” refers to conventional acid-addition salts orbase-addition salts that retain the biological effectiveness andproperties of the compounds of the present invention and are formed fromsuitable non-toxic organic or inorganic acids or organic or inorganicbases. Acid-addition salts include for example those derived frominorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, andthose derived from organic acids such as p-toluenesulfonic acid,salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citricacid, malic acid, lactic acid, fumaric acid, and the like. Base-additionsalts include those derived from ammonium, potassium, sodium and,quaternary ammonium hydroxides, such as for example, tetramethylammonium hydroxide. The chemical modification of a pharmaceuticalcompound into a salt is a technique well known to pharmaceuticalchemists in order to obtain improved physical and chemical stability,hygroscopicity, flowability and solubility of compounds. It is forexample described in Bastin, Organic Process Research & Development2000, 4, 427-435 or in Ansel, In: Pharmaceutical Dosage Forms and DrugDelivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example,the pharmaceutically acceptable salt of the compounds provided hereinmay be a sodium salt.

The pharmaceutical composition of the invention is formulated, dosed,and administered in a fashion consistent with good medical practice.Factors for consideration in this context include the particular mammalbeing treated, the clinical condition of the individual patient, thesite of delivery of the agent, the method of administration, thescheduling of administration, the age and sex of the patients and otherfactors known to medical practitioners. Herein, an “effective amount”(also known as “(therapeutically) effective dose”) means the amount of acompound that will elicit the biological or medical response of asubject that is being sought by a medical doctor or other clinician. The“effective amount” of the inhibitor of the invention, the combinedpreparation of the invention, or the pharmaceutical composition of theinvention will be governed by such considerations, and is the minimumamount necessary to inhibit HBsAg and/or HBeAg. For example, such amountmay be below the amount that is toxic to the cells of the recipient, orto the mammal as a whole.

For example, if the PAPD5 inhibitor or the PAPD7 inhibitor is/anantisense oligonucleotide, then the pharmaceutically effective amountadministered is a dose of 0.1-15 mg/kg, such as from 0.2-10 mg/kg, suchas from 0.25-5 mg/kg. The administration can be once a week, every2^(nd) week, every third week or even once a month.

The nucleic acid molecules or pharmaceutical compositions of the presentinvention may be administered topical (such as, to the skin, inhalation,ophthalmic or otic) or enteral (such as, orally or through thegastrointestinal tract) or parenteral (such as, intravenous,subcutaneous, intra-muscular, intracerebral, intracerebroventricular orintrathecal).

In a preferred embodiment the nucleic acid molecule or pharmaceuticalcompositions of the present invention are administered by a parenteralroute including intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion, intrathecal orintracranial, e.g. intracerebral or intraventricular, intravitrealadministration. In one embodiment the active oligonucleotide oroligonucleotide conjugate is administered intravenously. In anotherembodiment the active nucleic acid molecule or nucleic acid moleculeconjugate is administered subcutaneously.

The inhibitor of the invention, the combined preparation of theinvention, or the pharmaceutical composition of the invention is usefulin the prevention and/or treatment of an HBV invention. They preferablyinhibit secretion of HBsAg and/or HBeAg, most preferably of HBsAg andHBeAg.

Definitions

Nucleotide Sequence

The term “nucleotide sequence” or “polynucleotide” is commonly known inthe art and comprises molecules comprising or consisting of naturallyoccurring molecules such as DNA and RNA as well as nucleic acidanalogues such as, e.g., oligonucleotides thiophosphates, substitutedribo-oligonucleotides, LNA molecules, PNA molecules, GNA (glycol nucleicacid) molecules, TNA (threose nucleic acid) molecules, morpholinopolynucleotides, or nucleic acids with modified backbones such aspolysiloxane, and 2′-O-(2-methoxy) ethyl-phosphorothioate, or a nucleicacid with substituents, such as methyl-, thio-, sulphate, benzoyl-,phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or areporter molecule to facilitate its detection. Furthermore, the term“nucleotide sequence” is to be construed equivalently with the term“nucleic acid molecule” in context of the present invention and mayinter alia refer to DNA, RNA, PNA or LNA or hybrids thereof or anymodification thereof that is known in the art (see, e.g., U.S. Pat. Nos.5,525,711, 4,711,955, 5,792,608 or EP 302175 for examples ofmodifications). Nucleic acid residues comprised by the nucleic acidsequence described and provided herein may be naturally occurringnucleic acid residues or artificially produced nucleic acid residues.Examples for nucleic acid residues are adenine (A), guanine (G),cytosine (C), thymine (T), uracil (U), xanthine (X), and hypoxanthine(HX). As understood by the person of skill in the art, thymine (T) anduracil (U) may be used interchangeably depending on the respective typeof polynucleotide. For example, as the skilled person is aware of, athymine (T) as part of a DNA corresponds to an uracil (U) as part of thecorresponding transcribed mRNA. The polynucleotides described andprovided herein may be single- or double-stranded, linear or circular,natural or synthetic.

The nucleotide sequences provided herein may be cloned into a vector.The term “vector” as used herein includes plasmids, cosmids, viruses,bacteriophages and other vectors commonly used in genetic engineering.In a preferred embodiment, these vectors are suitable for thetransformation of cells, like mammalian cells or yeast cells. Herein,the vector may be an expression vector. Generally, expression vectorshave been widely described in the literature. They may comprise aselection marker gene and a replication-origin ensuring replication inthe host, a promoter, and a termination signal for transcription.Between the promoter and the termination signal there may be at leastone restriction site or a polylinker which enables the insertion of anucleic acid sequence desired to be expressed. Non-limiting examples forthe vector into which a nucleotide sequence provided herein may becloned are adenoviral, adeno-associated viral (AAV), lentiviral,HIV-based lentiviral, nonviral minicircle-vectors, or other vectors forbacterial and eukaryotic expression systems.

Nucleic Acid Molecule

The term “nucleic acid molecule” or “therapeutic nucleic acid molecule”as used herein is defined as it is generally understood by the skilledperson as a molecule comprising two or more covalently linkednucleosides (i.e. a nucleotide sequence). The nucleic acid molecule(s)referred to in the method of the invention are generally therapeuticoligonucleotides below 50 nucleotides in length. The nucleic acidmolecules may be or comprise an antisense oligonucleotide, or may beanother oligomeric nucleic acid molecule, such as a CRISPR RNA, a siRNA,shRNA, an aptamer, or a ribozyme. Nucleic acid molecules arecompositions that are commonly made in the laboratory by solid-phasechemical synthesis followed by purification. When referring to asequence of the nucleic acid molecule, reference is made to the sequenceor order of nucleobase moieties, or modifications thereof, of thecovalently linked nucleotides or nucleosides. The nucleic acid moleculeof the invention is man-made, and is chemically synthesized, and istypically purified or isolated. The nucleic acid molecule of theinvention may comprise one or more modified nucleosides or nucleotides.

In some embodiments, the nucleic acid molecule of the inventioncomprises or consists of 8 to 40 nucleotides in length, such as from 9to 35, such as from 10 to 30, such as from 11 to 22, such as from 12 to20, such as from 13 to 18 or 14 to 16 contiguous nucleotides in length.

In some embodiments, the nucleic acid molecule or contiguous nucleotidesequence thereof comprises or consists of 22 or less nucleotides, suchas 20 or less nucleotides, such as 18 or less nucleotides, such as 14,15, 16 or 17 nucleotides. It is to be understood that any range givenherein includes the range endpoints. Accordingly, if a nucleic acidmolecule is said to include from 10 to 30 nucleotides, both 10 and 30nucleotides are included.

In some embodiments, the contiguous nucleotide sequence comprises orconsists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides in length

The nucleic acid molecule(s) are typically for modulating the expressionof a target nucleic acids in a mammal. In some embodiments the nucleicacid molecules, such as for siRNAs, shRNAs and antisenseoligonucleotides, are typically for inhibiting the expression of atarget nucleic acid. The nucleic acid molecules, when combined, maytherefore be effective at modulating the expression of one or moretarget nucleic acids in a mammal.

In one embodiment of the invention the nucleic acid molecule is selectedfrom a RNAi agent, such as a siRNA, shRNA or an antisenseoligonucleotide. In preferred embodiments the nucleic acid molecule is ahigh affinity modified antisense oligonucleotide.

In some embodiments the nucleic acid molecule is a phosphorothioatenucleic acid molecule. In some embodiments the nucleic acid moleculecomprises phosphorothioate internucleoside linkages.

In some embodiments the nucleic acid molecule(s) may be conjugated tonon-nucleosidic moieties (conjugate moieties).

A library of nucleic acid molecules is to be understood as a collectionof variant nucleic acid molecules. The purpose of the library of nucleicacid molecules can vary. In some embodiments, the library of nucleicacid molecules is composed of oligonucleotides with different nucleobasesequences, for example it may be a library of nucleic acid moleculeswhich are designed across a target nucleic acid (e.g. a RNA sequence),for example a library of antisense oligonucleotides or siRNA moleculesmay be generated by a mRNA gene-walk with the purpose of identifyingregions on the target nucleic acid where nucleic acid moleculesefficiently modulate the target nucleic acid. In some embodiments, thelibrary of nucleic acid molecules is composed of oligonucleotides withoverlapping nucleobase sequence targeting a specific region on thetarget nucleic acid with the purpose of identifying the most potentsequence within the library of nucleic acid molecules. In someembodiments, the library of nucleic acid molecules is a library ofnucleic acid molecule design variants (child nucleic acid molecules) ofa parent or ancestral nucleic acid molecule, wherein the nucleic acidmolecule design variants retaining the core nucleobase sequence of theparent nucleic acid molecule.

Oligonucleotide

The term “oligonucleotide” as used herein is defined as it is generallyunderstood by the skilled person as a molecule comprising two or morecovalently linked nucleosides. Such covalently bound nucleosides mayalso be referred to as nucleic acid molecules or oligomers.Oligonucleotides are commonly made in the laboratory by solid-phasechemical synthesis followed by purification. When referring to asequence of the oligonucleotide, reference is made to the sequence ororder of nucleobase moieties, or modifications thereof, of thecovalently linked nucleotides or nucleosides. The oligonucleotide of theinvention is man-made, and is chemically synthesized, and is typicallypurified or isolated. The oligonucleotide of the invention may compriseone or more modified nucleosides or nucleotides. An antisenseoligonucleotide is a single stranded oligonucleotide with minimal or nointernal duplex formation. A siRNA molecule generally consists of 2complementary oligonucleotide stands (a sense strand and an antisensestrand) that forms a double stranded molecule. A shRNA molecule is anoligonucleotide which is generally longer than antisenseoligonucleotides and which form an internal duplex (hairpin) structurewithin the molecule.

Antisense Oligonucleotides

The term “Antisense oligonucleotide” as used herein is defined asoligonucleotides capable of modulating expression of a target gene byhybridizing to a target nucleic acid, in particular to a contiguoussequence on a target nucleic acid. The antisense oligonucleotides arenot essentially double stranded and are therefore not siRNAs or shRNAs.Preferably, the antisense oligonucleotides of the present invention aresingle stranded.

RNAi

Herein, the term “RNA interference (RNAi) molecule” refers to anymolecule inhibiting RNA expression or translation, including the nucleicacid molecules defined herein. A small interfering RNA (siRNA) is adouble-stranded RNA molecule that, by binding complementary mRNA aftertranscription, leads to their degradation and loss in translation. Asmall hairpin RNA (shRNA) is an artificial RNA molecule with a hairpinstructure which upon expression is able to reduce mRNA via the DICER andRNA reducing silencing complex (RISC). RNAi molecules can be designed onthe base of the RNA sequence of the gene of interest. Corresponding RNAican then be synthesized chemically or by in vitro transcription, orexpressed from a vector or PCR product

siRNA and shRNA molecules are generally between 20 and 50 nucleotides inlength, such as between 25 and 35 nucleotides in length, and interactswith the endonuclease known as Dicer which is believed to processesdsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs which are then incorporated into an RNA-inducedsilencing complex (RISC). Effective extended forms of Dicer substrateshave been described in U.S. Pat. Nos. 8,349,809 and US 8,513,207, herebyincorporated by reference. Upon binding to the appropriate target mRNA,one or more endonucleases within the RISC cleave the target to inducesilencing. RNAi agents may be chemically modified using modifiedinternucleotide linkages and high affinity nucleosides, such as 2′-4′bicyclic ribose modified nucleosides, including LNA and cET.

Contiguous Nucleotide Sequence

The term “contiguous nucleotide sequence” refers to the region of theoligonucleotide which is complementary to the target nucleic acid. Theterm is used interchangeably herein with the term “contiguous nucleobasesequence” and the term “oligonucleotide motif sequence”. In someembodiments all the nucleotides of the oligonucleotide constitute thecontiguous nucleotide sequence. In some embodiments the oligonucleotidecomprises the contiguous nucleotide sequence and may optionally comprisefurther nucleotide(s), for example a nucleotide linker region which maybe used to attach a functional group to the contiguous nucleotidesequence. The nucleotide linker region may or may not be complementaryto the target nucleic acid.

Nucleotides

Nucleotides are the building blocks of oligonucleotides andpolynucleotides, and for the purposes of the present invention includeboth naturally occurring and non-naturally occurring nucleotides. Innature, nucleotides, such as DNA and RNA nucleotides comprise a ribosesugar moiety, a nucleobase moiety and one or more phosphate groups(which is absent in nucleosides). Nucleosides and nucleotides may alsointerchangeably be referred to as “units” or “monomers”.

Modified Nucleoside

The term “modified nucleoside” or “nucleoside modification” as usedherein refers to nucleosides modified as compared to the equivalent DNAor RNA nucleoside by the introduction of one or more modifications ofthe sugar moiety or the (nucleo)base moiety. In a preferred embodimentthe modified nucleoside comprise a modified sugar moiety. The termmodified nucleoside may also be used herein interchangeably with theterm “nucleoside analogue” or modified “units” or modified “monomers”.Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA orRNA nucleosides herein. Nucleosides with modifications in the baseregion of the DNA or RNA nucleoside are still generally termed DNA orRNA if they allow Watson Crick base pairing.

Modified Internucleoside Linkage

The term “modified internucleoside linkage” is defined as generallyunderstood by the skilled person as linkages other than phosphodiester(PO) linkages, that covalently couples two nucleosides together.Nucleotides with modified internucleoside linkage are also termed“modified nucleotides”. In some embodiments, the modifiedinternucleoside linkage increases the nuclease resistance of the nucleicacid molecules of the invention compared to a phosphodiester linkage.For naturally occurring oligonucleotides, the internucleoside linkageincludes phosphate groups creating a phosphodiester bond betweenadjacent nucleosides. Modified internucleoside linkages are particularlyuseful in stabilizing oligonucleotides as well as siRNA's for in vivouse, and may serve to protect against nuclease cleavage at regions ofDNA or RNA nucleosides in the oligonucleotide or siRNA of the invention,for example within the gap region of a gapmer oligonucleotide, as wellas in regions of modified nucleosides.

In an embodiment, the nucleic acid molecule, e.g. antisenseoligonucleotide, shRNA or siRNA, comprises one or more internucleosidelinkages modified from the natural phosphodiester to a linkage that isfor example more resistant to nuclease attack. Nuclease resistance maybe determined by incubating the oligonucleotide in blood serum or byusing a nuclease resistance assay (e.g. snake venom phosphodiesterase(SVPD), both are well known in the art. Internucleoside linkages whichare capable of enhancing the nuclease resistance of an oligonucleotideare referred to as nuclease resistant internucleoside linkages. In someembodiments at least 50% of the internucleoside linkages in theantisense oligonucleotide, or contiguous nucleotide sequence thereof,are modified, such as at least 60%, such as at least 70%, such as atleast 80 or such as at least 90% of the internucleoside linkages in theoligonucleotide, or contiguous nucleotide sequence thereof, aremodified. In some embodiments all of the internucleoside linkages of theoligonucleotide, or contiguous nucleotide sequence thereof, aremodified. It will be recognized that, in some embodiments thenucleosides which link the oligonucleotide of the invention to anon-nucleotide functional group, such as a conjugate, may bephosphodiester. In some embodiments all of the internucleoside linkagesof the oligonucleotide, or contiguous nucleotide sequence thereof, arenuclease resistant internucleoside linkages.

Modified internucleoside linkages may be selected from the groupcomprising phosphorothioate, diphosphorothioate and boranophosphate. Insome embodiments, the modified internucleoside linkages are compatiblewith the RNaseH recruitment of the oligonucleotide of the invention, forexample phosphorothioate, diphosphorothioate or boranophosphate.

In some embodiments the internucleoside linkage comprises sulphur (S),such as a phosphorothioate internucleoside linkage.

A phosphorothioate internucleoside linkage is particularly useful due tonuclease resistance, beneficial pharmakokinetics and ease ofmanufacture. In some embodiments at least 50% of the internucleosidelinkages in the oligonucleotide, or contiguous nucleotide sequencethereof, are phosphorothioate, such as at least 60%, such as at least70%, such as at least 80 or such as at least 90% of the internucleosidelinkages in the oligonucleotide, or contiguous nucleotide sequencethereof, are phosphorothioate. In some embodiments all of theinternucleoside linkages of the oligonucleotide, or contiguousnucleotide sequence thereof, are phosphorothioate.

In some embodiments, the oligonucleotide comprises one or more neutralinternucleoside linkage, particularly a internucleoside linkage selectedfrom phosphotriester, methylphosphonate, MMI, amide-3, formacetal orthioformacetal.

Further internucleoside linkages are disclosed in WO2009/124238(incorporated herein by reference). In an embodiment the internucleosidelinkage is selected from linkers disclosed in WO2007/031091(incorporated herein by reference). Particularly, the internucleosidelinkage may be selected from —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—,—S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—,—S—P(O)₂—S—, —O—PO(R^(H))—O—, 0-PO(OCH₃)-0-, —O—PO(NR^(H))—O—,—O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—, —O—PO(NHR^(H))—O—, —O—P(O)₂NR^(H)—,—NR^(H)—P(O)₂O—, —NR^(H)—CO—NR^(H)—, and/or the internucleoside linkermay be selected form the group consisting of: —O—CO—O—, —O—CO—NR^(H)—,—NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—,—CH₂—NR^(H)CO—, —O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—,—CH₂—SO₂—CH₂—, —CH₂—CO—NR^(H)—, —O—CH₂ 1'CH₂—NR^(H)—CO—,—CH₂—NCH₃—O—CH₂—, where R^(H) is selected from hydrogen and C1-4-alkyl.

Nuclease resistant linkages, such as phosphothioate linkages, areparticularly useful in antisense oligonucleotide regions capable ofrecruiting nuclease when forming a duplex with the target nucleic acid,such as region G for gapmers, or the non-modified nucleoside region ofheadmers and tailmers. Phosphorothioate linkages may, however, also beuseful in non-nuclease recruiting regions and/or affinity enhancingregions such as regions F and F′ for gapmers, or the modified nucleosideregion of headmers and tailmers.

Each of the design regions may however comprise internucleoside linkagesother than phosphorothioate, such as phosphodiester linkages, inparticularly in regions where modified nucleosides, such as LNA, protectthe linkage against nuclease degradation. Inclusion of phosphodiesterlinkages, such as one or two linkages, particularly between or adjacentto modified nucleoside units (typically in the non-nuclease recruitingregions) can modify the bioavailability and/or bio-distribution of anoligonucleotide—see WO2008/113832, incorporated herein by reference.

In an embodiment all the internucleoside linkages in the antisenseoligonucleotide are phosphorothioate and/or boranophosphate linkages.Preferably, all the internucleoside linkages in the oligonucleotide arephosphorothioate linkages.

Nucleobase

The term nucleobase includes the purine (e.g. adenine and guanine) andpyrimidine (e.g. uracil, thymine and cytosine) moiety present innucleosides and nucleotides which form hydrogen bonds in nucleic acidhybridization. In the context of the present invention the termnucleobase also encompasses modified nucleobases which may differ fromnaturally occurring nucleobases, but are functional during nucleic acidhybridization. In this context “nucleobase” refers to both naturallyoccurring nucleobases such as adenine, guanine, cytosine, thymidine,uracil, xanthine and hypoxanthine, as well as non-naturally occurringvariants. Such variants are for example described in Hirao et al (2012)Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009)Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

In a some embodiments the nucleobase moiety is modified by changing thepurine or pyrimidine into a modified purine or pyrimidine, such assubstituted purine or substituted pyrimidine, such as a nucleobasedselected from isocytosine, pseudoisocytosine, 5-methyl cytosine,5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil,5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2′thio-thymine, inosine,diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and2-chloro-6-aminopurine.

The nucleobase moieties may be indicated by the letter code for eachcorresponding nucleobase, e.g. A, T, G, C or U, wherein each letter mayoptionally include modified nucleobases of equivalent function. Forexample, in the exemplified oligonucleotides, the nucleobase moietiesare selected from A, T, G, C, and 5-methyl cytosine. Optionally, for LNAgapmers, 5-methyl cytosine LNA nucleosides may be used.

Modified Oligonucleotide

The term modified oligonucleotide or modified nucleic acid moleculedescribes an oligonucleotide or nucleic acid molecule comprising one ormore sugar-modified nucleosides and/or modified internucleosidelinkages. The term “chimeric” is a term that has been used in theliterature to describe oligonucleotides or nucleic acid molecules withmodified nucleosides, in particular gapmer oligonucleotides.

Complementarity

The term “complementarity” describes the capacity for Watson-Crickbase-pairing of nucleosides/nucleotides. Watson-Crick base pairs areguanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U). It willbe understood that oligonucleotides may comprise nucleosides withmodified nucleobases, for example 5-methyl cytosine is often used inplace of cytosine, and as such the term complementarity encompassesWatson Crick base-paring between non-modified and modified nucleobases(see for example Hirao et al (2012) Accounts of Chemical Research vol.45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic AcidChemistry Suppl. 37 1.4.1).

The term “% complementary” as used herein, refers to the number ofnucleotides in percent of a contiguous nucleotide sequence in a nucleicacid molecule (e.g. oligonucleotide) which, at a given position, arecomplementary to (i.e. form Watson Crick base pairs with) a contiguousnucleotide sequence, at a given position of a separate nucleic acidmolecule (e.g. the target nucleic acid). The percentage is calculated bycounting the number of aligned bases that form pairs between the twosequences (when aligned with the target sequence 5′-3′ and theoligonucleotide sequence from 3′-5′), dividing by the total number ofnucleotides in the oligonucleotide and multiplying by 100. In such acomparison a nucleobase/nucleotide which does not align (form a basepair) is termed a mismatch. Preferably, insertions and deletions are notallowed in the calculation of % complementarity of a contiguousnucleotide sequence.

The term “fully complementary”, refers to 100% complementarity.

The following is an example of an oligonucleotide (SEQ ID NO: 12) thatis fully complementary to a region of a target nucleic acid (SEQ ID NO:10).

1651 cccttagttaaacacatctacccttgacca 1680 (Pos. 1651-1690 of SEQ ID NO: 10)          |||||||||||||||||||   1 --3′-TCAATTTGTGTAGATGGGA-5′---   19 (SEQ ID NO: 12)

Identity

In context of the present invention, the term “identity” or “percentidentity” means that amino acid or nucleotide sequences have identitiesof at least 80%, preferably at least 90%, more preferably at least 95%,even more preferably at least 98%, and even more preferably at least 99%identity to the sequences shown herein, e.g. those of SEQ ID NO: 1, 2,or 3, wherein the higher identity values are preferred upon the lowerones. In accordance with the present invention, the term“identity/identities” or “percent identity/identities” in the context oftwo or more nucleic acid or amino acid sequences, refers to two or moresequences that are the same, or that have a specified percentage ofamino acid residues or nucleotides that are the same (e.g., at least80%, at least 90%, at least 95%, at least 98%, or at least 99% identitywith the amino acid sequences of, e.g., SEQ ID NO: 1, 2, 3, 7, 8 or 9 orwith the nucleotide sequences of, e.g., SEQ ID NO: 4, 5, 6, 10 or 11),when compared and aligned for maximum correspondence over a window ofcomparison, or over a designated region as measured using a sequencecomparison algorithm as known in the art, or by manual alignment andvisual inspection.

For amino acid sequences, preferably the described identity exists overa region that is at least about 50 amino acids, preferably at least 100amino acids, more preferably at least 400 amino acids, more preferablyat least 500 amino acids, more preferably at least 600 amino acids andmost preferably all amino acids in length.

In case of nucleotide sequences, the described identity most preferablyexists over a region that is at least 100 nucleotides, preferably atleast 1,000 nucleotides, more preferably at least 2,000 nucleotides andmost preferably all nucleotides in length. However, for nucleic acidmolecules, which generally are below 50 nucleotides, the identity can beassessed over a significantly shorter region. Generally, the percentageidentity of nucleic acid molecules is calculated by counting the numberof aligned bases that are identical between the two sequences dividingby the total number of nucleotides in the nucleic acid molecule andmultiplying by 100. Percent Identity=(Matches×100)/Length of alignedregion. Preferably, insertions and deletions are not allowed in thecalculation of % identity of a contiguous nucleotide sequence in anucleic acid molecule.

Those having skills in the art will know how to determine percentidentity between/among sequences using, for example, algorithms such asthose based on CLUSTALW computer program (Thompson, 1994, Nucl AcidsRes, 2: 4673-4680) or FASTDB (Brutlag, 1990, Comp App Biosci, 6:237-245), as known in the art. Also available to those having skills inthis art are the BLAST and BLAST 2.0 algorithms (Altschul, 1997, NuclAcids Res 25: 3389-3402; Altschul, 1993, J Mol Evol, 36: 290-300;Altschul, 1990, J Mol Biol 215: 403-410). For example, BLAST 2.0, whichstands for Basic Local Alignment Search Tool BLAST (Altschul, 1997, loc.cit.; Altschul, 1993, loc. cit.; Altschul, 1990, loc. cit.), can be usedto search for local sequence alignments. BLAST, as discussed above,produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying similar sequences.

Analogous computer techniques using BLAST (Altschul, 1997, loc. cit.;Altschul, 1993, loc. cit.; Altschul, 1990, loc. cit.) are used to searchfor identical or related molecules in nucleotide databases such asGenBank or EMBL.

Hybridization

The term “hybridizing” or “hybridizes” as used herein is to beunderstood as two nucleic acid strands (e.g. an oligonucleotide and atarget nucleic acid) forming hydrogen bonds between base pairs onopposite strands thereby forming a duplex. The affinity of the bindingbetween two nucleic acid strands is the strength of the hybridization.It is often described in terms of the melting temperature (T_(m))defined as the temperature at which half of the oligonucleotides areduplexed with the target nucleic acid. At physiological conditions T_(m)is not strictly proportional to the affinity (Mergny and Lacroix, 2003,Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG°is a more accurate representation of binding affinity and is related tothe dissociation constant (K_(d)) of the reaction by ΔG°=−RTIn(K_(d)),where R is the gas constant and T is the absolute temperature.Therefore, a very low ΔG° of the reaction between an oligonucleotide andthe target nucleic acid reflects a strong hybridization between theoligonucleotide and target nucleic acid. ΔG° is the energy associatedwith a reaction where aqueous concentrations are 1M, the pH is 7, andthe temperature is 37° C. The hybridization of oligonucleotides to atarget nucleic acid is a spontaneous reaction and for spontaneousreactions ΔG° is less than zero. ΔG° can be measured experimentally, forexample, by use of the isothermal titration calorimetry (ITC) method asdescribed in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al.,2005, Drug Discov Today. The skilled person will know that commercialequipment is available for ΔG° measurements. ΔG° can also be estimatednumerically by using the nearest neighbor model as described bySantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 usingappropriately derived thermodynamic parameters described by Sugimoto etal., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004,Biochemistry 43:5388-5405. In order to have the possibility ofmodulating its intended nucleic acid target by hybridization,oligonucleotides of the present invention hybridize to a target nucleicacid with estimated ΔG° values below −10 kcal for oligonucleotides thatare 10-30 nucleotides in length. In some embodiments the degree orstrength of hybridization is measured by the standard state Gibbs freeenergy ΔG°. The oligonucleotides may hybridize to a target nucleic acidwith estimated ΔG° values below the range of −10 kcal, such as below −15kcal, such as below −20 kcal and such as below −25 kcal foroligonucleotides that are 8-30 nucleotides in length. In someembodiments the oligonucleotides hybridize to a target nucleic acid withan estimated ΔG° value of −10 to −60 kcal, such as −12 to −40, such asfrom −15 to −30 kcal or −16 to −27 kcal such as −18 to −25 kcal.

Target Nucleic Acid

According to the present invention, the target nucleic acid is a nucleicacid which encodes mammalian PAPD5 or PAPD7 and may for example be agene, a RNA, a mRNA, and pre-mRNA, a mature mRNA or a cDNA sequence. Thetarget may therefore be referred to as a PAPD5 or PAPD7 target nucleicacid. The oligonucleotide or nucleic acid molecule of the invention mayfor example target exon regions of a mammalian PAPD5 or PAPD7, or mayfor example target intron region in the PAPD5 or PAPD7 pre-mRNA.Suitably, the target nucleic acid encodes a PAPD5 or PAPD7 protein, inparticular mammalian PAPD5 or PAPD7, such as human PAPD5 or PAPD7 (Seefor example tables 1A and B and Table 2A and B) which provides the mRNAand pre-mRNA sequences for human, monkey, rat and pig PAPD5 or PAPD7).

In some embodiments, the target nucleic acid is selected from the groupconsisting of SEQ ID NO: 4, 5 or 10 or naturally occurring variantsthereof (e.g. sequences encoding a mammalian PAPD5).

In some embodiments, the target nucleic acid is selected from the groupconsisting of SEQ ID NO: 6 or 11 or naturally occurring variants thereof(e.g. sequences encoding a mammalian PAPD7).

If employing the oligonucleotide of the invention in research ordiagnostics the target nucleic acid may be a cDNA or a synthetic nucleicacid derived from DNA or RNA.

For in vivo or in vitro application, the oligonucleotide of theinvention is typically capable of inhibiting the expression of the PAPD5or PAPD7 target nucleic acid in a cell which is expressing the PAPD5 orPAPD7 target nucleic acid. The contiguous sequence of nucleobases of theoligonucleotide of the invention is typically complementary to the PAPD5or PAPD7 target nucleic acid, as measured across the length of theoligonucleotide, optionally with the exception of one or two mismatches,and optionally excluding nucleotide based linker regions which may linkthe oligonucleotide to an optional functional group such as a conjugate,or other non-complementary terminal nucleotides (e.g. region D′ or D″).The target nucleic acid may, in some embodiments, be a RNA or DNA, suchas a messenger RNA, such as a mature mRNA or a pre-mRNA. In someembodiments the target nucleic acid is a RNA or DNA which encodesmammalian PAPD5 or PAPD7 protein, such as human PAPD5 or PAPD7, e.g. thehuman PAPD5 mRNA sequence, such as that disclosed as SEQ ID NO 4, 5 or10 or the human PAPD7 mRNA sequence, such as that disclosed as SEQ ID NO6 or 11. Further information on exemplary target nucleic acids isprovided in tables 1A and B and Table 2A and B.

TABLE 1A Genome and assembly information for PAPD5 across species.Genomic coordinates Species Chr. Band Strand Start End ensembl_gene_idAssembly Human 16 q12.1 fwd 50152918 50235310 ENSG00000121274 GRCh38.p7mouse 8 C3 fwd 88199213 88259722 ENSMUSG00000036779 GRCm38.p5 Rat 19 p11rev 19771677 19832812 ENSRNOG00000024212 Rnor_6.0

TABLE 1B Genome and assembly information for PAPD7 across species.Genomic coordinates Species Chr Band Strand Start End ensembl_gene_idAssembly Human 5 p15.31 fwd 6713007 6757048 ENSG00000112941 GRCh38.p7mouse 13 B3 rev 69497959 69534617 ENSMUSG00000034575 GRCm38.p5 Rat 1 p11fwd 36400443 36433238 ENSRNOG00000017613 Rnor_6.0 Fwd = forward strand.Rev = reverse strand. The genome coordinates provide the pre-mRNAsequence (genomic sequence).

Target Sequence

The term “target sequence” as used herein refers to a sequence ofnucleotides present in the target nucleic acid which comprises thenucleobase sequence which is complementary to the oligonucleotide ornucleic acid molecule of the invention. In some embodiments, the targetsequence consists of a region on the target nucleic acid which iscomplementary to the contiguous nucleotide sequence of theoligonucleotide of the invention (i.e. a sub-sequence).

The oligonucleotide or nucleic acid molecule of the invention comprisesa contiguous nucleotide sequence which is complementary to or hybridizesto a region on the target nucleic acid, such as a target sequencedescribed herein.

The target nucleic sequence to which the oligonucleotide iscomplementary to or hybridizes to generally comprises a stretch ofcontiguous nucleobases of at least 10 nucleotides. The contiguousnucleotide sequence is between 10 to 50 nucleotides, such as 12-30, suchas 13 to 25, such as 14 to 20, such as 15 to 18 contiguous nucleotides.

Naturally Occurring Variant

The term “naturally occurring variant” refers to variants of PAPD5 orPAPD7 gene or transcripts which originate from the same genetic loci asthe target nucleic acid, but may differ for example, by virtue ofdegeneracy of the genetic code causing a multiplicity of codons encodingthe same amino acid, or due to alternative splicing of pre-mRNA, or thepresence of polymorphisms, such as single nucleotide polymorphisms, andallelic variants. Based on the presence of the sufficient complementarysequence to the oligonucleotide, the oligonucleotide of the inventionmay therefore target the target nucleic acid and naturally occurringvariants thereof.

In some embodiments, the naturally occurring variants have at least 95%such as at least 98% or at least 99% homology to a mammalian PAPD5target nucleic acid, such as a target nucleic acid selected form thegroup consisting of SEQ ID NO: 4, 5 or 10.

In some embodiments, the naturally occurring variants have at least 95%such as at least 98% or at least 99% homology to a mammalian PAPD5target nucleic acid, such as a target nucleic acid selected form thegroup consisting of SEQ ID NO: 6 or 11.

Numerous single nucleotide polymorphisms are known in the PAPD5 or PAPD7gene, for example those disclosed in Table 2A (human premRNAstart/reference sequence is SEQ ID NO: 10) and Table 2B human premRNAstart/reference sequence is SEQ ID NO: 11).

TABLE 2A PAPD5 polymorphisms (naturally occurring variants) minor alleleMinor allele frequency Start on SEQ ID NO: 10 G 0.00399361 29 G0.000199681 34 T 0.000399361 39 A 0.000599042 62 A 0.000599042 97 G0.000199681 141 A 0.000199681 142 T 0.000199681 158 A 0.0241613 235 A0.00239617 279 — 0.214058 370 G 0.000798722 450 CAGCA 0.000798722 603 A0.0223642 1028 C 0.000199681 1044 A 0.0189696 1068 T 0.000199681 1181 T0.0249601 1199 T 0.000998403 1258 A 0.000199681 1261 T 0.000599042 1441T 0.000199681 1443 C 0.000599042 1469 A 0.000399361 1535

TABLE 2B PAPD7 polymorphisms (naturally occurring variants) Minor Startminor allele allele frequency on SEQ ID NO: 11 A 0.293331 21 T0.00119808 50 T 0.000199681 64 A 0.00279553 127 A 0.0597045 224 G0.000199681 234 T 0.000599042 270 A 0.128994 284 C 0.000399361 316 T0.000199681 349 G 0.00778754 362 A 0.000199681 409 G 0.000199681 425 A0.000199681 448 T 0.000199681 473 C 0.000199681 491 C 0.327676 564 T0.0203674 606 — 0.389577 837 — 0.00139776 1317 T 0.000599042 1331 T0.000199681 1475 T 0.000399361 1483 C 0.01877 1673 A 0.000199681 1682 T0.00339457 1726 GGTCCTGGCCGGCGCCCGC 0.258586 1736 G 0.000599042 1760 C0.000199681 1777 G 0.000399361 1780 T 0.000199681 1852 T 0.0001996811861 T 0.000199681 1889 C 0.000399361 1923 G 0.000399361 1962 T0.0147764 1987 G 0.000998403 1996 T 0.000399361 2036

Modulation of Expression

The term “modulation of expression” as used herein is to be understoodas an overall term for a nucleic acid molecules ability to alter theamount of PAPD5 or PAPD7 when compared to the amount of PAPD5 or PAPD7before administration of the nucleic acid molecule. Alternativelymodulation of expression may be determined by reference to a controlexperiment. It is generally understood that the control is an individualor target cell treated with a saline composition or an individual ortarget cell treated with a non-targeting or nucleic acid molecule(mock). It may however also be an individual treated with the standardof care.

One type of modulation is a nucleic acid molecules ability to inhibit,down-regulate, reduce, suppress, remove, stop, block, prevent, lessen,lower, avoid or terminate expression of PAPD5 or PAPD7, e.g. bydegradation of mRNA or blockage of transcription.

High Affinity Modified Nucleosides

A high affinity modified nucleoside is a modified nucleotide which, whenincorporated into the oligonucleotide enhances the affinity of theoligonucleotide for its complementary target, for example as measured bythe melting temperature (T^(m)). A high affinity modified nucleoside ofthe present invention preferably result in an increase in meltingtemperature between +0.5 to +12° C., more preferably between +1.5 to+10° C. and most preferably between+3 to +8° C. per modified nucleoside.Numerous high affinity modified nucleosides are known in the art andinclude for example, many 2′ substituted nucleosides as well as lockednucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997,25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000,3(2), 293-213).

Sugar Modifications

The nucleic acid molecule of the invention may comprise one or morenucleosides which have a modified sugar moiety, i.e. a modification ofthe sugar moiety when compared to the ribose sugar moiety found in DNAand RNA.

Numerous nucleosides with modification of the ribose sugar moiety havebeen made, primarily with the aim of improving certain properties ofnucleic acid molecules, such as affinity and/or nuclease resistance.

Such modifications include those where the ribose ring structure ismodified, e.g. by replacement with a hexose ring (HNA), or a bicyclicring, which typically have a biradicle bridge between the C2 and C4carbons on the ribose ring (LNA), or an unlinked ribose ring whichtypically lacks a bond between the C2 and C3 carbons (e.g. UNA). Othersugar modified nucleosides include, for example, bicyclohexose nucleicacids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798).Modified nucleosides also include nucleosides where the sugar moiety isreplaced with a non-sugar moiety, for example in the case of peptidenucleic acids (PNA), or morpholino nucleic acids.

Sugar modifications also include modifications made via altering thesubstituent groups on the ribose ring to groups other than hydrogen, orthe 2′-OH group naturally found in DNA and RNA nucleosides. Substituentsmay, for example be introduced at the 2′, 3′, 4′ or 5′ positions.Nucleosides with modified sugar moieties also include 2′ modifiednucleosides, such as 2′ substituted nucleosides. Indeed, much focus hasbeen spent on developing 2′ substituted nucleosides, and numerous 2′substituted nucleosides have been found to have beneficial propertieswhen incorporated into oligonucleotides, such as enhanced nucleosideresistance and enhanced affinity.

2′ Modified Nucleosides.

A 2′ sugar modified nucleoside is a nucleoside which has a substituentother than H or —OH at the 2′ position (2′ substituted nucleoside) orcomprises a 2′ linked biradicle, and includes 2′ substituted nucleosidesand LNA (2′-4′ biradicle bridged) nucleosides. For example, the 2′modified sugar may provide enhanced binding affinity and/or increasednuclease resistance to the oligonucleotide. Examples of 2′ substitutedmodified nucleosides are 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA,2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA, and 2′-F-ANAnucleoside. For further examples, please see e.g. Freier & Altmann;Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in DrugDevelopment, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry andBiology 2012, 19, 937. Below are illustrations of some 2′ substitutedmodified nucleosides.

Locked Nucleic Acid Nucleosides (LNA).

LNA nucleosides are modified nucleosides which comprise a linker group(referred to as a biradicle or a bridge) between C2′ and C4′ of theribose sugar ring of a nucleotide. These nucleosides are also termedbridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.

In some embodiments, the modified nucleoside or the LNA nucleosides ofthe oligomer of the invention has a general structure of the formula Ior II:

wherein W is selected from —O—, —S—, —N(R^(a))—, —C(R^(a)R^(b))—, suchas, in some embodiments —O—;

B designates a nucleobase or modified nucleobase moiety;

Z designates an internucleoside linkage to an adjacent nucleoside, or a5-terminal group;

Z* designates an internucleoside linkage to an adjacent nucleoside, or a3′-terminal group;

X designates a group selected from the list consisting of—C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—, —C(R^(a))═N—, —O—, —Si(R^(a))₂—,—S—, —SO₂—, —N(R^(a))—, and >C═Z

In some embodiments, X is selected from the group consisting of: —O—,—S—, NH—, NR^(a)R^(b), —CH₂—, CR^(a)R^(b), —C(═CH₂)—, and—C(═CR^(a)R^(b))—

In some embodiments, X is —O—

Y designates a group selected from the group consisting of—C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—, —C(R^(a))═N—, —O—, —S—, —SO₂—,—N(R^(a))—, and >C═Z

In some embodiments, Y is selected from the group consisting of: —CH₂—,—C(R^(a)R^(b))—, —CH₂CH₂—, —C(R^(a)R^(b))—C(R^(a)R^(b))—, —CH₂CH₂CH₂—,—C(R^(a)R^(b))C(R^(a)R^(b))C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—, and—C(R^(a))═N—

In some embodiments, Y is selected from the group consisting of: —CH₂—,—CHR^(a)—, —CHCH₃—, CR^(a)R^(b)—

or —X—Y— together designate a bivalent linker group (also referred to asa radicle) together designate a bivalent linker group consisting of 1,2, 3 or 4 groups/atoms selected from the group consisting of—C(R^(a)R^(b))—, —C(R^(a))=C(R^(b))—, —C(R^(a))═N—, —O—, —Si(R^(a))₂—,—S—, —SO₂—, —N(R^(a))—, and >C═Z,

In some embodiments, —X—Y— designates a biradicle selected from thegroups consisting of: —X—CH₂—, —X—CR^(a)R^(b)—, —X—C(HCH₃), —O—Y—,—O—CH₂—, —S—CH₂—, —N—CH₂—, —O—CHCH₃—, —CH₂—O—CH₂, —O—CH(CH₃CH₃)—,—O—CH₂—CH₂—, OCH₂—CH₂—CH₂—, —O——CH₂OCH₂—, —O—NCH₂—, —O(═CH₂)—CH₂—,—NR^(a)—CH₂—, N—O—CH₂, —S—CR^(a)R^(b)— and —S—CHR^(a)—.

In some embodiments —X—Y— designates —O—CH₂— or —O—CH(CH₃)—.

wherein Z is selected from —O—, —S—, and —N(R^(a))—,

and R^(a) and, when present R^(b), each is independently selected fromhydrogen, optionally substituted C₁₋₆-alkyl, optionally substitutedC₂₋₆-alkenyl, optionally substituted C₂₋₆-alkynyl, hydroxy, optionallysubstituted C₁₋₆-alkoxy, C₂₋₅-alkoxyalkyl, C₂₋₆-alkenyloxy, carboxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl,aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl,heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino,carbamoyl, mono- and di(C₁₋₆-alkyl)-amino-carbonyl,amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, where aryl and heteroaryl maybe optionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂),wherein for all chiral centers, asymmetric groups may be found in eitherR or S orientation.

wherein R¹, R², R³, R⁵ and R^(5*) are independently selected from thegroup consisting of: hydrogen, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substitutedC₂₋₆-alkynyl, hydroxy, C₁₋₆-alkoxy, C₂₋₆-alkoxyalkyl, C₂₋₆-alkenyloxy,carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆ alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, where aryl and heteroaryl maybe optionally substituted, and where two geminal substituents togethermay designate oxo, thioxo, imino, or optionally substituted methylene.

In some embodiments R¹, R², R³, R⁵ and R^(5*) are independently selectedfrom C₁₋₆ alkyl, such as methyl, and hydrogen.

In some embodiments R¹, R², R³, R⁵ and R^(5*) are all hydrogen.

In some embodiments R¹, R², R³, are all hydrogen, and either R⁵ andR^(5*) is also hydrogen and the other of R⁵ and R^(5*)is other thanhydrogen, such as C₁₋₆ alkyl such as methyl.

In some embodiments, R^(a) is either hydrogen or methyl. In someembodiments, when present, R^(b) is either hydrogen or methyl.

In some embodiments, one or both of R^(a) and R^(b) is hydrogen

In some embodiments, one of R^(a) and R^(b) is hydrogen and the other isother than hydrogen

In some embodiments, one of R^(a) and R^(b) is methyl and the other ishydrogen

In some embodiments, both of R^(a) and R^(b) are methyl.

In some embodiments, the biradicle —X—Y— is —O—CH₂—, W is O, and all ofR¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such LNA nucleosides aredisclosed in WO99/014226, WO00/66604, WO98/039352 and WO2004/046160which are all hereby incorporated by reference, and include what arecommonly known as beta-D-oxy LNA and alpha-L-oxy LNA nucleosides.

In some embodiments, the biradicle —X—Y— is —S—CH₂—, W is O, and all ofR¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such thio LNA nucleosidesare disclosed in WO99/014226 and WO2004/046160 which are herebyincorporated by reference.

In some embodiments, the biradicle —X—Y— is —NH—CH₂—, W is O, and all ofR¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such amino LNA nucleosidesare disclosed in W099/014226 and WO2004/046160 which are herebyincorporated by reference.

In some embodiments, the biradicle —X—Y— is —O—CH₂—CH₂— or—O—CH₂—CH₂—CH₂—, W is O, and all of R¹, R², R³, R⁵ and R^(5*) are allhydrogen. Such LNA nucleosides are disclosed in WO00/047599 and Moritaet al, Bioorganic & Med.Chem. Lett. 12 73-76, which are herebyincorporated by reference, and include what are commonly known as2′-O-4′C-ethylene bridged nucleic acids (ENA).

In some embodiments, the biradicle —X—Y— is —O—CH₂—, W is O, and all ofR¹, R², R³, and one of R⁵ and R^(5*) are hydrogen, and the other of R⁵and R^(5*) is other than hydrogen such as C₁₋₆ alkyl, such as methyl.Such 5′ substituted LNA nucleosides are disclosed in WO2007/134181 whichis hereby incorporated by reference.

In some embodiments, the biradicle —X—Y— is —O—CR^(a)R^(b)—, wherein oneor both of R^(a) and R^(b) are other than hydrogen, such as methyl, W isO, and all of R¹, R², R³, and one of R⁵ and R5* are hydrogen, and theother of R⁵ and R^(5*) is other than hydrogen such as C₁₋₆ alkyl, suchas methyl. Such bis modified LNA nucleosides are disclosed inWO2010/077578 which is hereby incorporated by reference.

In some embodiments, the biradicle —X—Y— designate the bivalent linkergroup —O—CH(CH₂OCH₃)— (2′ O-methoxyethyl bicyclic nucleic acid—Seth atal., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81). In some embodiments,the biradicle —X—Y— designate the bivalent linker group —O—CH(CH₂CH₃)—(2′0-ethyl bicyclic nucleic acid—Seth at al., 2010, J. Org. Chem. Vol75(5) pp. 1569-81). In some embodiments, the biradicle —X—Y— is—O—CHR^(a)—, W is O, and all of R¹, R², R³, R⁵ and R^(5*) are allhydrogen. Such 6′ substituted LNA nucleosides are disclosed inWO10036698 and WO07090071 which are both hereby incorporated byreference.

In some embodiments, the biradicle —X—Y— is —O—CH(CH₂OCH₃)—, W is O, andall of R¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such LNA nucleosidesare also known as cyclic MOEs in the art (cMOE) and are disclosed inWO07090071.

In some embodiments, the biradicle —X—Y— designate the bivalent linkergroup —O—CH(CH₃)—. —in either the R- or S-configuration. In someembodiments, the biradicle —X—Y— together designate the bivalent linkergroup —O—CH₂—O—CH₂— (Seth at al., 2010, J. Org. Chem). In someembodiments, the biradicle —X—Y— is —O—CH(CH₃)—, W is O, and all of R¹,R², R³, R⁵ and R^(5*) are all hydrogen. Such 6′ methyl LNA nucleosidesare also known as cET nucleosides in the art, and may be either (S)cETor (R)cET stereoisomers, as disclosed in WO07090071 (beta-D) andWO2010/036698 (alpha-L) which are both hereby incorporated byreference).

In some embodiments, the biradicle —X—Y— is —O—CR^(a)R^(b)—, wherein inneither R^(a) or R^(b) is hydrogen, W is O, and all of R¹, R², R³, R⁵and R^(5*) are all hydrogen. In some embodiments, R^(a) and R^(b) areboth methyl. Such 6′ di-substituted LNA nucleosides are disclosed in WO2009006478 which is hereby incorporated by reference.

In some embodiments, the biradicle —X—Y— is —S—CHR^(a)—, W is O, and allof R¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such 6′ substituted thioLNA nucleosides are disclosed in WO11156202 which is hereby incorporatedby reference. In some 6′ substituted thio LNA embodiments R^(a) ismethyl.

In some embodiments, the biradicle —X—Y— is —C(═CH₂)—C(R^(a)R^(b))—,such as —C(═CH₂)—CH₂—, or —C(═CH₂)—CH(CH₃)—W is O, and all of R¹, R²,R³, R⁵ and R^(5*) are all hydrogen. Such vinyl carbo LNA nucleosides aredisclosed in WO08154401 and WO09067647 which are both herebyincorporated by reference.

In some embodiments the biradicle —X—Y— is —N(—OR^(a))—, W is O, and allof R¹, R², R³, R⁵ and R^(5*) are all hydrogen. In some embodiments R^(a)is C₁₋₆ alkyl such as methyl. Such LNA nucleosides are also known as Nsubstituted LNAs and are disclosed in WO2008/150729 which is herebyincorporated by reference. In some embodiments, the biradicle —X—Y—together designate the bivalent linker group —O—NR^(a)—CH₃— (Seth atal., 2010, J. Org. Chem). In some embodiments the biradicle —X—Y— is—N(R^(a))—, W is O, and all of R¹, R², R³, R⁵ and R^(5*) are allhydrogen. In some embodiments R^(a) is C₁₋₆ alkyl such as methyl.

In some embodiments, one or both of R⁵ and R^(5*) is hydrogen and, whensubstituted the other of R⁵ and R^(5*) is C₁₋₆ alkyl such as methyl. Insuch an embodiment, R¹, R², R³, may all be hydrogen, and the biradicle—X—Y— may be selected from —O—CH₂— or —O—C(HCR^(a))—, such as—O—C(HCH₃)—.

In some embodiments, the biradicle is —CR^(a)R^(b)—O—CR^(a)R^(b)—, suchas CH₂—O—CH₂—, W is O and all of R¹, R², R³, R⁵ and R^(5*) are allhydrogen. In some embodiments R^(a) is C₁₋₆ alkyl such as methyl. SuchLNA nucleosides are also known as conformationally restrictednucleotides (CRNs) and are disclosed in WO2013036868 which is herebyincorporated by reference.

In some embodiments, the biradicle is —O—CR^(a)R^(b)—O—CR^(a)R^(b)—,such as O—CH₂—O—CH₂—, W is O and all of R¹, R², R³, R⁵ and R^(5*) areall hydrogen. In some embodiments R^(a) is C₁₋₆alkyl such as methyl.Such LNA nucleosides are also known as COC nucleotides and are disclosedin Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238, whichis hereby incorporated by reference.

It will be recognized than, unless specified, the LNA nucleosides may bein the beta-D or alpha-L stereoisoform.

Certain examples of LNA nucleosides are presented in Scheme 1.

As illustrated in the examples, in some embodiments of the invention theLNA nucleosides in the oligonucleotides are beta-D-oxy-LNA nucleosides.

Nuclease Mediated Degradation

Nuclease mediated degradation refers to an oligonucleotide capable ofmediating degradation of a complementary nucleotide sequence whenforming a duplex with such a sequence.

In some embodiments, the oligonucleotide may function via nucleasemediated degradation of the target nucleic acid, where theoligonucleotides of the invention are capable of recruiting a nuclease,particularly and endonuclease, preferably endoribonuclease (RNase), suchas RNase H. Examples of oligonucleotide designs which operate vianuclease mediated mechanisms are oligonucleotides which typicallycomprise a region of at least 5 or 6 DNA nucleosides and are flanked onone side or both sides by affinity enhancing nucleosides, for examplegapmers, headmers and tailmers.

RNase H Activity and Recruitment

The RNase H activity of an antisense oligonucleotide refers to itsability to recruit RNase H when in a duplex with a complementary RNAmolecule. WO01/23613 provides in vitro methods for determining RNaseHactivity, which may be used to determine the ability to recruit RNaseH.Typically an oligonucleotide is deemed capable of recruiting RNase H ifit, when provided with a complementary target nucleic acid sequence, hasan initial rate, as measured in pmol/l/min, of at least 5%, such as atleast 10% or more than 20% of the of the initial rate determined whenusing a oligonucleotide having the same base sequence as the modifiedoligonucleotide being tested, but containing only DNA monomers withphosphorothioate linkages between all monomers in the oligonucleotide,and using the methodology provided by Example 91-95 of WO01/23613(hereby incorporated by reference).

Gapmer

The term gapmer as used herein refers to an antisense oligonucleotidewhich comprises a region of RNase H recruiting oligonucleotides (gap)which is flanked 5′ and 3′ by regions which comprise one or moreaffinity enhancing modified nucleosides (flanks or wings). Variousgapmer designs are described herein. Headmers and tailmers areoligonucleotides capable of recruiting RNase H where one of the flanksis missing, i.e. only one of the ends of the oligonucleotide comprisesaffinity enhancing modified nucleosides. For headmers the 3′ flank ismissing (i.e. the 5′ flank comprises affinity enhancing modifiednucleosides) and for tailmers the 5′ flank is missing (i.e. the 3′ flankcomprises affinity enhancing modified nucleosides).

LNA Gapmer

The term LNA gapmer is a gapmer oligonucleotide wherein at least one ofthe affinity enhancing modified nucleosides is an LNA nucleoside.

Mixed Wing Gapmer

The term mixed wing gapmer or mixed flank gapmer refers to a LNA gapmerwherein at least one of the flank regions comprise at least one LNAnucleoside and at least one non-LNA modified nucleoside, such as atleast one 2′ substituted modified nucleoside, such as, for example,2′-O-alkyl-IRNA, 2′-O-methyl-IRNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA(MOE), 2′-amino-DNA, 2′-Fluoro-RNA and 2′-F-ANA nucleoside(s). In someembodiments the mixed wing gapmer has one flank which comprises only LNAnucleosides (e.g. 5′ or 3′) and the other flank (3′ or 5′ respectfully)comprises 2′ substituted modified nucleoside(s) and optionally LNAnucleosides.

Conjugate

The term conjugate as used herein refers to an oligonucleotide which iscovalently linked to a non-nucleotide moiety (conjugate moiety or regionC or third region).

Conjugation of the oligonucleotide of the invention to one or morenon-nucleotide moieties may improve the pharmacology of theoligonucleotide, e.g. by affecting the activity, cellular distribution,cellular uptake or stability of the oligonucleotide. In some embodimentsthe conjugate moiety modify or enhance the pharmacokinetic properties ofthe oligonucleotide by improving cellular distribution, bioavailability,metabolism, excretion, permeability, and/or cellular uptake of theoligonucleotide. In particular the conjugate may target theoligonucleotide to a specific organ, tissue or cell type and therebyenhance the effectiveness of the oligonucleotide in that organ, tissueor cell type. A the same time the conjugate may serve to reduce activityof the oligonucleotide in non-target cell types, tissues or organs, e.g.off target activity or activity in non-target cell types, tissues ororgans. WO 93/07883 and WO2013/033230 provides suitable conjugatemoieties, which are hereby incorporated by reference. Further suitableconjugate moieties are those capable of binding to theasialoglycoprotein receptor (ASGPr). In particular tri-valentN-acetylgalactosamine conjugate moieties are suitable for binding to thethe ASGPr, see for example WO 2014/076196, WO 2014/207232 and WO2014/179620 (hereby incorporated by reference).

Oligonucleotide conjugates and their synthesis has also been reported incomprehensive reviews by Manoharan in Antisense Drug Technology,Principles, Strategies, and Applications, S. T. Crooke, ed., Ch. 16,Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid DrugDevelopment, 2002, 12, 103, each of which is incorporated herein byreference in its entirety.

In an embodiment, the non-nucleotide moiety (conjugate moiety) isselected from the group consisting of carbohydrates, cell surfacereceptor ligands, drug substances, hormones, lipophilic substances,polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins,viral proteins (e.g. capsids) or combinations thereof.

Conjugate Linkers

A linkage or linker is a connection between two atoms that links onechemical group or segment of interest to another chemical group orsegment of interest via one or more covalent bonds. Conjugate moietiescan be attached to the oligonucleotide directly or through a linkingmoiety (e.g. linker or tether). Linkers serve to covalently connect athird region, e.g. a conjugate moiety to an oligonucleotide (e.g. thetermini of region A or C).

In some embodiments of the invention the conjugate or oligonucleotideconjugate of the invention may optionally, comprise a linker regionwhich is positioned between the oligonucleotide and the conjugatemoiety. In some embodiments, the linker between the conjugate andoligonucleotide is biocleavable.

Biocleavable linkers comprising or consisting of a physiologicallylabile bond that is cleavable under conditions normally encountered oranalogous to those encountered within a mammalian body. Conditions underwhich physiologically labile linkers undergo chemical transformation(e.g., cleavage) include chemical conditions such as pH, temperature,oxidative or reductive conditions or agents, and salt concentrationfound in or analogous to those encountered in mammalian cells. Mammalianintracellular conditions also include the presence of enzymatic activitynormally present in a mammalian cell such as from proteolytic enzymes orhydrolytic enzymes or nucleases. In one embodiment the biocleavablelinker is susceptible to S1 nuclease cleavage. In a preferred embodimentthe nuclease susceptible linker comprises between 1 and 10 nucleosides,such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, more preferablybetween 2 and 6 nucleosides and most preferably between 2 and 4 linkednucleosides comprising at least two consecutive phosphodiester linkages,such as at least 3 or 4 or 5 consecutive phosphodiester linkages.Preferably the nucleosides are DNA or RNA. Phosphodiester containingbiocleavable linkers are described in more detail in WO 2014/076195(hereby incorporated by reference).

Conjugates may also be linked to the oligonucleotide via nonbiocleavable linkers, or in some embodiments the conjugate may comprisea non-cleavable linker which is covalently attached to the biocleavablelinker. Linkers that are not necessarily biocleavable but primarilyserve to covalently connect a conjugate moiety to an oligonucleotide orbiocleavable linker. Such linkers may comprise a chain structure or anoligomer of repeating units such as ethylene glycol, amino acid units oramino alkyl groups. In some embodiments the linker (region Y) is anamino alkyl, such as a C₂-C₃₆ amino alkyl group, including, for exampleC₆ to C₁₂ amino alkyl groups. In some embodiments the linker (region Y)is a C₆ amino alkyl group. Conjugate linker groups may be routinelyattached to an oligonucleotide via use of an amino modifiedoligonucleotide, and an activated ester group on the conjugate group.

Treatment

The terms “treatment”, “treating”, “treats” or the like are used hereinto generally mean obtaining a desired pharmacological and/orphysiological effect. This effect is therapeutic in terms of partiallyor completely curing a disease and/or adverse effect attributed to thedisease. The term “treatment” as used herein covers any treatment of adisease in a subject and includes: (a) inhibiting the disease, i.e.arresting its development like the inhibition of increase of HBsAgand/or HBeAg; or (b) ameliorating (i.e. relieving) the disease, i.e.causing regression of the disease, like the repression of HBsAg and/orHBeAg production . Thus, a compound that ameliorates and/or inhibits aHBV infection is a compound that treats a HBV invention. Preferably, theterm “treatment” as used herein relates to medical intervention of analready manifested disorder, like the treatment of an already definedand manifested HBV infection.

Prevention

Herein the term “preventing”, “prevention” or “prevents” relates to aprophylactic treatment, i.e. to a measure or procedure the purpose ofwhich is to prevent, rather than to cure a disease. Prevention meansthat a desired pharmacological and/or physiological effect is obtainedthat is prophylactic in terms of completely or partially preventing adisease or symptom thereof. Accordingly, herein “preventing a HBVinfection” includes preventing a HBV infection from occurring in asubject, and preventing the occurrence of symptoms of a HBV infection.In the present invention in particular the prevention of HBV infectionin children from HBV infected mothers are contemplated.

Patient

For the purposes of the present invention the “subject” (or “patient”)may be a vertebrate. In context of the present invention, the term“subject” includes both humans and other animals, particularly mammals,and other organisms. Thus, the herein provided means and methods areapplicable to both human therapy and veterinary applications.Accordingly, herein the subject may be an animal such as a mouse, rat,hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovinespecies, horse, camel, or primate. Preferably, the subject is a mammal.More preferably the subject is human.

HBV Infection

The term “hepatitis B virus infection” or “HBV infection” is commonlyknown in the art and refers to an infectious disease that is caused bythe hepatitis B virus (HBV) and affects the liver. A HBV infection canbe an acute or a chronic infection. Some infected persons have nosymptoms during the initial infection and some develop a rapid onset ofsickness with vomiting, yellowish skin, tiredness, dark urine andabdominal pain (“Hepatitis B Fact sheet N° 204”. who.int. July 2014.Retrieved 4 November 2014). Often these symptoms last a few weeks andcan result in death. It may take 30 to 180 days for symptoms to begin.In those who get infected around the time of birth 90% develop a chronichepatitis B infection while less than 10% of those infected after theage of five do (“Hepatitis B FAQs for the Public—Transmission”, U.S.Centers for Disease Control and Prevention (CDC), retrieved 2011-11-29).Most of those with chronic disease have no symptoms; however, cirrhosisand liver cancer may eventually develop (Chang, 2007, Semin FetalNeonatal Med, 12: 160-167). These complications result in the death of15 to 25% of those with chronic disease (“Hepatitis B Fact sheet N°204”. who.int. July 2014, retrieved 4 Nov. 2014). Herein, the term “HBVinfection” includes the acute and chronic hepatitis B infection. Theterm “HBV infection” also includes the asymptotic stage of the initialinfection, the symptomatic stages, as well as the asymptotic chronicstage of the HBV infection.

Enzymatically Active Fragments

Herein, an enzymatically active fragment of SEQ ID NO: 1 or 2 (i.e. ofPAPD5) relates to those polypeptides that comprise a stretch ofcontiguous amino acid residues of SEQ ID NO: 1 or 2 (i.e. of PAPD5) andthat retain a biological activity (i.e. functionality) of PAPD5,particularly the poly-A polymerase function. In line with this, herein,an enzymatically active fragment of SEQ ID NO: 3 (i.e. of PAPD7) relatesto those polypeptides that comprise a stretch of contiguous amino acidresidues of SEQ ID NO: 3 (i.e. of PAPD7) and that retain a biologicalactivity (i.e. functionality) of PAPD7, particularly the poly-Apolymerase function. Examples for enzymatically active fragments ofPAPD5 and PAPD7 are the nucleotidyltransferase domain and the Cid1 polyA polymerase.

Polypeptide

Herein, term “polypeptide” includes all molecules that comprise orconsist of amino acid monomers linked by peptide (amide) bonds. Thus,the term “polypeptide” comprises all amino acid sequences, such aspeptides, oliogopeptides, polypeptides and proteins. The “polypeptide”described herein may be a naturally occurring polypeptide or anon-naturally occurring polypeptide. The non-naturally occurringpolypeptide may comprise at least one mutation (e.g. amino acidsubstitution, amino acid deletion or amino acid addition) as compared tothe naturally occurring counterpart. The non-naturally occurringpolypeptide may also be cloned in a vector and/or be operable linked toa promoter that is not the natural promoter of said polypeptide. Saidpromoter may be a constitutively active promoter. The term “amino acid”or “residue” as used herein includes both L- and D-isomers of thenaturally occurring amino acids as well as of other amino acids (e.g.,non-naturally-occurring amino acids, amino acids which are not encodedby nucleic acid sequences, synthetic amino acids etc.). Examples ofnaturally-occurring amino acids are alanine (Ala; A), arginine (Arg; R),asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C),glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine(His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K),methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine(Ser; S), threonine (Thr; T), tryptophane (Trp; W), tyrosine (Tyr; Y),valine (Val; V). Post-translationally modified naturally-occurring aminoacids are dehydrobutyrine (Dhb) and labionin (Lab). Examples fornon-naturally occurring amino acids are described above. Thenon-naturally occurring polypeptide may comprise one or more non-aminoacid substituents, or heterologous amino acid substituents, compared tothe amino acid sequence of a naturally occurring form of thepolypeptide, for example a reporter molecule or another ligand,covalently or non-covalently bound to the amino acid sequence.

Compound

Herein, the term “compound” means any nucleic acid molecule, such asRNAi molecules according to the invention or any conjugate comprisingsuch a nucleic acid molecule. For example, herein the compound may be anRNAi molecule against PAPD5 or PAPD7, in particular an antisenseoligonucleotide, a siRNA or a shRNA.

Composition

The term “composition” may also be used to describe a nucleic acidmolecule compound. A nucleic acid molecule composition has less than 20%impurities, preferably less than 15% or 10% impurities, more preferablyless than 9, 8, 7 or 6% impurities, most preferably less than 5%impurities. The impurities are typically nucleic acid molecules whichare one or two nucleotides shorter (n-1 or n-2) than the primary nucleicacid molecule component.

Inhibitor

The term “inhibitor” is known in the art and relates to acompound/substance or composition capable of fully or partiallypreventing or reducing the physiologic function (i.e. the activity) of(a) specific protein(s) (e.g. of PAPD5 and/or PAPD7). In the context ofthe present invention, an “inhibitor” of PAPD5 or PAPD7 is capable ofpreventing or reducing the activity/function of PAPD5 or PAPD7,respectively, by preventing or reducing the expression of the PAPD5 orPAPD7 gene products. Thus, an inhibitor of PAPD5 or PAPD7 may lead to adecreased expression level of PAPD5 or PAPD7 (e.g. decreased level ofPAPD5 or PAPD7 mRNA, or of PAPD5 or PAPD7 protein) which is reflected ina decreased functionality (i.e. activity) of PAPD5 or PAPD7, whereinsaid function comprises the poly-A polymerase function. An inhibitor ofPAPD5 or PAPD7, in the context of the present invention, accordingly,may also encompass transcriptional repressors of PAPD5 or PAPD7expression that are capable of reducing the level of PAPD5 or PAPD7.Preferred inhibitors are nucleic acid molecules of the invention.

Measuring

Herein, the term “measuring” also means “analyzing” or “determining”(i.e. detecting and/or quantifying). For example, the term “measuringthe expression and/or activity of PAPD5 and/or PAPD7” means determiningthe amount of PAPD5 and/or PAPD7 expression and/or activity, forexample, determining the amount of the PAPD5 and/or PAPD7 polypeptide(i.e. protein). Methods for measuring (i.e. determining) the amountand/or activity of PAPD5 and/or PAPD7 protein are known in the art anddescribed herein above. In line with this, the term “measuring whether atest compound inhibits propagation of HBV” means analyzing ordetermining (i.e. detecting and/or quantifying) whether a test compoundor composition inhibits propagation of HBV.

The present invention is further described by reference to thenon-limiting figures and examples.

EXAMPLES

The Examples illustrate the invention.

Material and Methods

Oligonucleotide Motif Sequences and Oligonucleotide Compounds

TABLE 3 list of oligonucleotide motif sequences (indicated by SEQ ID NO)targeting the human PAPD5 transcript (SEQ ID NO: 10), designsof these, as well as specific antisense oligonucleotide compounds(indicated by CMP ID NO) designed based on the motif sequence. SEQ CMPStart ID Oligonucleotide ID ID NO Motif Sequence Design Compound NONO: 10 dG 12 agggtagatgtgtttaact 2-14-3 AGggtagatgtgtttaACT  12_1 1656−22 13 cagcctaaacttagtgg 3-12-2 CAGcctaaacttagtGG  13_1 2000 −21 14aagccctcaatgtaaaacac 2-14-4 AAgccctcaatgtaaaACAC  14_1 2446 −22 15aatagcaagtagaggagag 3-13-3 AATagcaagtagaggaGAG  15_1 3059 −21 16aaataaggatactggcga 2-12-4 AAataaggatactgGCGA  16_1 4036 −21 17gagggaacacataataaaag 4-14-2 GAGGgaacacataataaaAG  17_1 4484 −20 18gtaatacctctcacattc 2-12-4 GTaatacctctcacATTC  18_1 5928 −21 19agtaacaccaatctcattg 2-13-4 AGtaacaccaatctcATTG  19_1 6652 −21 20gtgacagtattcaatgatc 2-13-4 GTgacagtattcaatGATC  20_1 7330 −22 21cagttccgtatcaccaac 2-13-3 CAgttccgtatcaccAAC  21_1 7702 −22 22aagtctaactcaaagccatc 2-15-3 AAgtctaactcaaagccATC  22_1 8292 −21 23aggcttccattttattgaa 2-14-3 AGgcttccattttattGAA  23_1 8625 −22 24ttttagaaaacgaggcta 2-12-4 TTttagaaaacgagGCTA  24_1 9866 −20 25gtattcttattcttgct 3-12-2 GTAttcttattcttgCT  25_1 10254 −20 26attattcccacagtaaga 4-12-2 ATTAttcccacagtaaGA  26_1 10881 −22 27aacaacaaacaggatgggc 3-14-2 AACaacaaacaggatggGC  27_1 11370 −21 28atatccacaatattctgat 4-13-2 ATATccacaatattctgAT  28_1 11790 −20 29aaagaaataatgtcgtctgg 2-14-4 AAagaaataatgtcgtCTGG  29_1 12413 −20 30ccagagtaaacaaatcc 3-11-3 CCAgagtaaacaaaTCC  30_1 12718 −21 31attcaacatttttagtcacc 2-15-3 ATtcaacatttttagtcACC  31_1 13555 −21 32tttggtaattctttttttag 3-14-3 TTTggtaattcttttttTAG  32_1 14297 −20 33caatgaggaaacaagagtca 2-14-4 CAatgaggaaacaagaGTCA  33_1 15137 −22 34ttcaaaataatgtgggaggt 3-14-3 TTCaaaataatgtgggaGGT  34_1 15695 −22 35ggatatttgatacggcaaat 2-15-3 GGatatttgatacggcaAAT  35_1 16145 −20 36ctataagaagcaaaccc 3-11-3 CTAtaagaagcaaaCCC  36_1 16714 −21 37atataattcacgtttcactt 3-14-3 ATAtaattcacgtttcaCTT  37_1 17097 −21 38aatgattcacatgaaggtta 3-14-3 AATgattcacatgaaggTTA  38_1 17420 −20 39gttaggattttgctatg 2-11-4 GTtaggattttgcTATG  39_1 18299 −20 40gtacaaatatcaaccgtat 3-13-3 GTAcaaatatcaaccgTAT  40_1 18669 −21 41cacactatttcaagatgcta 3-15-2 CACactatttcaagatgcTA  41_1 19681 −22 42cacctatacaatggagtatt 3-14-3 CACctatacaatggagtATT  42_1 20352 −22 43atcatacgtcattagagaac 2-14-4 ATcatacgtcattagaGAAC  43_1 20721 −21 44cagaacagatactttgcca 2-15-2 CAgaacagatactttgcCA  44_1 21111 −22 45aagaatggttggttaaggg 2-14-3 AAgaatggttggttaaGGG  45_1 21782 −21 46agaattggtaaactggactg 4-14-2 AGAAttggtaaactggacTG  46_1 22378 −22 47agaattatattggctgg 3-11-3 AGAattatattggcTGG  47_1 23160 −20 48cctaaaccagacagaaaaga 2-15-3 CCtaaaccagacagaaaAGA  48_1 23993 −22 49accaattagagcagaaatc 4-13-2 ACCAattagagcagaaaTC  49_1 24813 −21 50ttctaaataacagatgggtc 3-14-3 TTCtaaataacagatggGTC  50_1 25047 −21 51tttataatttttttccatct 3-14-3 TTTataatttttttccaTCT  51_1 26080 −20 52gcaaatatcagattaacctc 4-14-2 GCAAatatcagattaaccTC  52_1 26625 −22 53aacggtatggcagaagacaa 3-14-3 AACggtatggcagaagaCAA  53_1 26973 −22 54ttcaacctttactgcat 4-11-2 TTCAacctttactgcAT  54_1 27813 −20 55actgataaagggcatttcaa 2-14-4 ACtgataaagggcattTCAA  55_1 28357 −22 56cagtaggaatgtggctt 2-12-3 CAgtaggaatgtggCTT  56_1 28718 −21 57ttttatggcagggtttcac 3-14-2 TTTtatggcagggtttcAC  57_1 29327 −21 58tcactgttaaacctcac 2-11-4 TCactgttaaaccTCAC  58_1 29902 −20 59caattttctaattcaatggt 4-14-2 CAATtttctaattcaatgGT  59_1 30704 −20 60aagatataattcacccact 4-13-2 AAGAtataattcacccaCT  60_1 31008 −22 61gccacataaaggataaagt 2-13-4 GCcacataaaggataAAGT  61_1 31348 −22 62cccattagaagtaaggtga 2-15-2 CCcattagaagtaaggtGA  62_1 32367 −22 63atgtaaattaaaacttccc 2-13-4 ATgtaaattaaaactTCCC  63_1 32632 −21 64tgagagcataaaagtacgga 3-15-2 TGAgagcataaaagtacgGA  64_1 32945 −22 65ttcacaacaggtaaaggg 4-12-2 TTCAcaacaggtaaagGG  65_1 33593 −21 66tgcattcctaagtaacataa 2-14-4 TGcattcctaagtaacATAA  66_1 34801 −21 67agagaaaagtgatgagggaa 3-14-3 AGAgaaaagtgatgaggGAA  67_1 35368 −22 68atacggatcaccagctaaa 3-14-2 ATAcggatcaccagctaAA  68_1 36131 −21 69catgttatgcacagaagat 2-13-4 CAtgttatgcacagaAGAT  69_1 36712 −22 70cgctgaagaactaagtatta 2-14-4 CGctgaagaactaagtATTA  70_1 37282 −20 71caaacagatggtggtgata 3-14-2 CAAacagatggtggtgaTA  71_1 37870 −20 72agtagccattaggatg 3-11-2 AGTagccattaggaTG  72_1 38478 −20 73atacacaggctccataata 2-14-3 ATacacaggctccataATA  73_1 39639 −22 74gatttttgtatagtccacaa 3-15-2 GATUttgtatagtccacAA  74_1 40178 −21 75gcatctataaaaaagggaca 2-14-4 GCatctataaaaaaggGACA  75_1 41042 −22 76agtgcaagtatcgct 2-10-3 AGtgcaagtatcGCT  76_1 41734 −20 77ccaaaagaatcaagttcgta 3-15-2 CCAaaagaatcaagttcgTA  77_1 42442 −21 78cctcagaccaaatttattt 2-13-4 CCtcagaccaaatttATTT  78_1 43203 −22 79ttcaacaagcatctattgta 3-14-3 TTCaacaagcatctattGTA  79_1 43663 −22 80caaaggttgttgtactct 3-12-3 CAAaggttgttgtacTCT  80_1 44220 −21 81tcataaatctttttccacg 3-14-2 TCAtaaatctttttccaCG  81_1 44756 −20 82cttgttacggatttaatgtg 2-15-3 CTtgttacggatttaatGTG  82_1 45042 −21 83gctataaaaatagaagcc 3-12-3 GCTataaaaatagaaGCC  83_1 46202 −22 84tccttagcaaactaaacat 3-13-3 TCCttagcaaactaaaCAT  84_1 47142 −22 85agcaaaaggcaggtattcaa 3-15-2 AGCaaaaggcaggtattcAA  85_1 47843 −22 86gaatccatttacatattcac 3-14-3 GAAtccatttacatattCAC  86_1 48267 −21 87tccagtatccaaaacatac 2-13-4 TCcagtatccaaaacATAC  87_1 49256 −21 88agcttaaagaagaacggtt 4-13-2 AGCTtaaagaagaacggTT  88_1 49688 −22 89cacaacgtgcctacctt 2-13-2 CAcaacgtgcctaccTT  89_1 50508 −22 90ccagaatccaagaaaatgg 3-14-2 CCAgaatccaagaaaatGG  90_1 50764 −21 91tcacctcgaactaaacaagt 2-16-2 TCacctcgaactaaacaaGT  91_1 51561 −21 92gtatctttctgtactatt 2-12-4 GTatctttctgtacTATT  92_1 52461 −21 93gtcattctactaacaaacg 4-13-2 GTCAttctactaacaaaCG  93_1 53305 −21 94tggaaaaggaagaaccatt 2-13-4 TGgaaaaggaagaacCATT  94_1 53865 −20 95aatacaactcttccgtgat 2-13-4 AAtacaactcttccgTGAT  95_1 54638 −22 96aataccctgacgagctg 2-12-3 AAtaccctgacgagCTG  96_1 54942 −22 97tcataaaacatgatccttgc 3-15-2 TCAtaaaacatgatccttGC  97_1 55741 −22 98ctaaagcagatccatagaa 3-13-3 CTAaagcagatccataGAA  98_1 56277 −21 99agactataacttttgctaca 3-15-2 AGActataacttttgctaCA  99_1 56942 −22 100agcaatgacttgaacatagt 2-16-2 AGcaatgacttgaacataGT 100_1 57369 −20 101ataaaacaagcatacgggc 3-14-2 ATAaaacaagcatacggGC 101_1 58146 −21 102atgagataccagcagatag 2-15-2 ATgagataccagcagatAG 102_1 58588 −20 103agaagaaatcctgagtaatc 4-14-2 AGAAgaaatcctgagtaaTC 103_1 59089 −21 104ccctaaaaagtgacgta 3-12-2 CCCtaaaaagtgacgTA 104_1 59461 −21 105ttaagttagatcacggc 4-11-2 TTAAgttagatcacgGC 105_1 59970 −20 106gtggatacagaaagcca 2-12-3 GTggatacagaaagCCA 106_1 60738 −22 107gtatcggcaggagatt 3-10-3 GTAtcggcaggagATT 107_1 61038 −21 108taactaattgattccattgc 4-14-2 TAACtaattgattccattGC 108_1 61868 −22 109agaagaacggaaattgcc 3-13-2 AGAagaacggaaattgCC 109_1 62418 −21 110ataatgattttcctatcc 4-12-2 ATAAtgattttcctatCC 110_1 62822 −20 111atggttttgtggagaagg 2-13-3 ATggttttgtggagaAGG 111_1 63000 −22 112tgctgctgtgaaaagaaatg 2-14-4 TGctgctgtgaaaagaAATG 112_1 63697 −21 113gtgtccaattttttattat 2-14-3 GTgtccaattttttatTAT 113_1 64377 −20 114gatggaatcaactgtgtagt 2-16-2 GAtggaatcaactgtgtaGT 114_1 65307 −21 115gatggtgacaaattattct 4-13-2 GATGgtgacaaattattCT 115_1 65894 −22 116tgcttttgggaatcttt 2-11-4 TGcttttgggaatCTTT 116_1 66650 −21 117gatgtcctacaatgaacacg 2-15-3 GAtgtcctacaatgaacACG 117_1 68024 −22 118gtacaaggacaaagtaacc 3-13-3 GTAcaaggacaaagtaACC 118_1 68732 −22 119tgaaacgcctatctcta 4-11-2 TGAAacgcctatctcTA 119_1 69029 −21 120atactatttatgcttatgga 3-15-2 ATActatttatgcttatgGA 120_1 69796 −21 121ttgtaatcaaggcaataagg 3-14-3 TTGtaatcaaggcaataAGG 121_1 70770 −21 122gaagtccaataacgcaga 4-12-2 GAAGtccaataacgcaGA 122_1 71091 −22 123atatccaatctctatatgtg 3-15-2 ATAtccaatctctatatgTG 123_1 72013 −21 124gccttacacaagactatatt 2-15-3 GCcttacacaagactatATT 124_1 73444 −22 125tgctgaattttatgttaac 4-13-2 TGCTgaattttatgttaAC 125_1 73823 −21 126cacaagatgatgggtttaag 2-14-4 CAcaagatgatgggttTAAG 126_1 74559 −22 127ttagtggtttgggtgc 2-12-2 TTagtggtttgggtGC 127_1 75043 −20 128agattgttaccttactgat 2-14-3 AG attgttaccttactGAT 128_1 76110 −21 129tattacaaatatcaatctcc 3-14-3 TATtacaaatatcaatcTCC 129_1 76931 −21 130taccaaaagcatagagtgg 3-14-2 TACcaaaagcatagagtGG 130_1 77605 −21 131aattatcttcccgctac 2-11-4 AAttatcttcccgCTAC 131_1 78652 −22 Motifsequences represent the contiguous sequence of nucleobases present inthe oligonucleotide.

Designs refer to the gapmer design, F-G-F′, where each number representsthe number of consecutive modified nucleosides, e.g. 2′ modifiednucleosides (first number=5′ flank), followed by the number of DNAnucleosides (second number=gap region), followed by the number ofmodified nucleosides, e.g2′ modified nucleosides (third number=3′flank), optionally preceded by or followed by further repeated regionsof DNA and LNA, which are not necessarily part of the contiguoussequence that is complementary to the target nucleic acid.

TABLE 4 list of oligonucleotide motif sequences (indicated by SEQ ID NO)targeting the human PAPD7 transcript (SEQ ID NO: 11), designsof these, as well as specific antisense oligonucleotide compounds(indicated by CMP ID NO) designed based on the motif sequence. SEQ StartID Oligonucleotide CMP ID NO: NO motif sequence Design Compound ID NO 11dG 132 gttgggtggaataggca 2-13-2 GTtgggtggaataggCA 132_1   132 −22 133tcgatttcccgttccaa 2-13-2 TCgatttcccgttccAA 133_1  1962 −21 134acaacctacacataaattgc 3-15-2 ACAacctacacataaattGC 134_1  2510 −21 135actataagaactcccaaca 2-13-4 ACtataagaactcccAACA 135_1  2668 −21 136gagaaaaagagttacaagc 4-13-2 GAGAaaaagagttacaaGC 136_1  2695 −20 137aactggagggagagaagag 4-13-2 AACTggagggagagaagAG 137_1  2883 −22 138tttctaagagcagaggtaca 2-15-3 TTtctaagagcagaggtACA 138_1  3090 −22 139agaagtaacaagagcct 4-11-2 AGAAgtaacaagagcCT 139_1  3463 −20 140agtatcaaaccagacctc 2-13-3 AGtatcaaaccagacCTC 140_1  3795 −22 141ccacaaccgaaagactt 4-11-2 CCACaaccgaaagacTT 141_1  4205 −22 142aatacacactgcattttca 2-13-4 AAtacacactgcattTTCA 142_1  4336 −20 143ccaggtagatagcacag 2-13-2 CCaggtagatagcacAG 143_1  4686 −21 144ccatgacaaagtaacaacag 2-14-4 CCatgacaaagtaacaACAG 144_1  4821 −22 145cagaatttcctttgagtta 2-14-3 CAg aatttcctttg agTTA 145_1  5134 −21 146ccttcgcaagaaagaattga 2-16-2 CCttcgcaagaaagaattGA 146_1  5263 −21 147tcatacatacacgcttct 2-14-2 TCatacatacacgcttCT 147_1  5577 −20 148tgcgaaaagattggagg 2-11-4 TGcgaaaagattgGAGG 148_1  5945 −21 149cacaggacgcttacatgaat 2-16-2 CAcaggacgcttacatgaAT 149_1  6235 −22 150gctgtttttttttcttaac 3-14-2 GCTgtttttttttcttaAC 150_1  6352 −21 151accataagtgagtgttctt 2-14-3 ACcataagtgagtgttCTT 151_1  6834 −22 152acacaagcccatagaaacag 2-16-2 ACacaagcccatagaaacAG 152_1  7158 −21 153cagtagtaaccaccaag 2-11-4 CAgtagtaaccacCAAG 153_1  7447 −22 154cctgcaaacttttatttat 2-14-3 CCtgcaaacttttattTAT 154_1  7708 −21 155acttagtaatagcagca 2-12-3 ACttagtaatagcaGCA 155_1  8074 −20 156atgaatactccgaagactt 2-13-4 ATgaatactccgaagACTT 156_1  8249 −21 157aaagaaaaggatcacaagcc 3-14-3 AAAgaaaaggatcacaaGCC 157_1  8784 −22 158agacagaaatcacctaaca 3-14-2 AGAcagaaatcacctaaCA 158_1  8887 −20 159tagaacagacattattcatc 3-14-3 TAGaacagacattattcATC 159_1  9506 −20 160agttacacggagcagcac 2-14-2 AGttacacggagcagcAC 160_1  9664 −22 161cactatacacagaacactat 3-14-3 CACtatacacagaacacTAT 161_1  9770 −22 162agctgtctaaatacatgg 2-12-4 AG ctgtctaaatacATGG 162_1 10000 −21 163atgaacctattttatgcttc 3-14-3 ATGaacctattttatgcTTC 163_1 10206 −22 164accatcattaacctgcgt 2-14-2 ACcatcattaacctgcGT 164_1 10318 −22 165agtaaagtgcccagatgt 2-14-2 AGtaaagtgcccagatGT 165_1 10568 −22 166ttccctatgaaatcctcaa 3-14-2 TTCcctatgaaatcctcAA 166_1 10781 −21 167cactcttcatagaatgcaac 2-14-4 CActcttcatagaatgCAAC 167_1 10917 −22 168aatgcttaatttttctctct 2-14-4 AAtgcttaatttttctCTCT 168_1 11084 −22 169ttagagacgatgcctataac 3-14-3 TTAgagacgatgcctatAAC 169_1 11308 −22 170tgaatagttcccatagatt 4-13-2 TGAAtagttcccatagaTT 170_1 11585 −21 171cagcataattgttttcttt 3-13-3 CAGcataattgttttcTTT 171_1 12330 −21 172atgtcattatgttttagtt 4-13-2 ATGTcattatgttttagTT 172_1 12634 −21 173cagcagtatctcttagaa 2-13-3 CAgcagtatctcttaGAA 173_1 12902 −21 174cggtaagggttcggtg 2-12-2 CGgtaagggttcggTG 174_1 13126 −21 175catgaaccacattaggaac 4-13-2 CATGaaccacattaggaAC 175_1 13383 −21 176cattcaacacacacgacaa 2-13-4 CAttcaacacacacgACAA 176_1 13578 −21 177aagtatccaagactcaaga 2-13-4 AAgtatccaagactcAAGA 177_1 13889 −20 178ccacagaaacaccgag 4-10-2 CCACagaaacaccgAG 178_1 14100 −22 179tggaaaagggaagggaaga 3-14-2 TGGaaaagggaagggaaGA 179_1 14179 −21 180agagagtccgaagcctg 2-13-2 AGagagtccgaagccTG 180_1 14432 −22 181atgggaaaggtaacgagc 3-13-2 ATGggaaaggtaacgaGC 181_1 14616 −22 182ctatcctacaagtccgaa 3-13-2 CTAtcctacaagtccgAA 182_1 15471 −22 183cattgcttttataatccta 4-13-2 CATTgcttttataatccTA 183_1 15816 −22 184ctttttaaggacaggagg 4-12-2 CTTTttaaggacaggaGG 184_1 15988 −21 185gatgaaagataagtgagcat 2-14-4 GAtgaaagataagtgaGCAT 185_1 16395 −22 186gaagcctgtaataattaagc 2-14-4 GAagcctgtaataattAAGC 186_1 17007 −22 187caccctagtaaagcaaac 4-12-2 CACCctagtaaagcaaAC 187_1 17151 −22 188gcaaatgtaagccttttt 3-13-2 GCAaatgtaagcctttTT 188_1 17303 −21 189acctgacagctaccgac 2-13-2 ACctgacagctaccgAC 189_1 17498 −22 190aagagtgggttgtaagc 2-12-3 AAgagtgggttgtaAGC 190_1 17963 −20 191tagtgaaaatatttggagtt 2-14-4 TAgtgaaaatatttggAGTT 191_1 18101 −20 192tttcagcaccttaaaccc 2-14-2 TTtcagcaccttaaacCC 192_1 18518 −22 193ttaagggaaaggaaacgtca 4-14-2 TTAAgggaaaggaaacgtCA 193_1 18747 −21 194gtaggtaaagggcaaaggaa 3-15-2 GTAggtaaagggcaaaggAA 194_1 19007 −22 195gtgaattaaagccaaagc 2-12-4 GTgaattaaagccaAAGC 195_1 19252 −21 196tgtttttgtattttagtat 3-13-3 TGTUttgtattttagTAT 196_1 19476 −20 197gaggttttttttagtgaatt 2-14-4 GAggttttttttagtgAATT 197_1 19722 −21 198gaggagctaaacggaca 3-12-2 GAGgagctaaacggaCA 198_1 20062 −22 199gtttagtcttatgttctcac 2-16-2 GTttagtcttatgttctcAC 199_1 20623 −21 200caaatactgaatatgcccg 2-14-3 CAaatactgaatatgcCCG 200_1 20726 −22 201accatttaaatcgccaac 3-12-3 ACCatttaaatcgccAAC 201_1 20926 −21 202cagtaagagtagcccaacaa 2-16-2 CAgtaagagtagcccaacAA 202_1 21234 −22 203taacggcaacatcaaatagc 4-14-2 TAACggcaacatcaaataGC 203_1 22017 −22 204actgagcaccaactacac 2-13-3 ACtgagcaccaactaCAC 204_1 22592 −22 205cctaattttatgtatcacat 2-14-4 CCtaattttatgtatcACAT 205_1 23074 −22 206ctaggaattataacaaatca 4-14-2 CTAGgaattataacaaatCA 206_1  23462, −20 23509207 actccaaagaacatactcac 2-14-4 ACtccaaagaacatacTCAC 207_1 24628 −22 208taagagaaacaatcacacca 3-14-3 TAAgagaaacaatcacaCCA 208_1 24935 −22 209tgtcctaaaatatcagcag 2-14-3 TGtcctaaaatatcagCAG 209_1 26022 −21 210cttttaatgagacagtgca 2-15-2 CTtttaatgagacagtgCA 210 1 26212 −20 211ttgtagcataagatggaaag 4-14-2 TTGTagcataagatggaaAG 211 1 26500 −21 212aaactgtagccaataactgt 3-14-3 AAActgtagccaataacTGT 212 1 26945 −21 213attcatcctaacacaagtag 2-15-3 ATtcatcctaacacaagTAG 213 1 27117 −22 214cacgaaaggaacagctaag 4-13-2 CACGaaaggaacagctaAG 214 1 27264 −21 215acaacaggcaagtacc 4-10-2 ACAAcaggcaagtaCC 215 1 27411 −20 216gctaaacactataaggat 3-12-3 GCTaaacactataagGAT 216 1 27505 −21 217cccgtaagcatttgagaa 2-14-2 CCcgtaagcatttgagAA 217 1 27831 −21 218agccaatatgcgacagtaac 2-16-2 AGccaatatgcgacagtaAC 218 1 28146 −22 219taaccaaaacaatcagtgtc 3-14-3 TAAccaaaacaatcagtGTC 219 1 28777 −20 220aacagggaacaggagtta 3-12-3 AACagggaacaggagTTA 220_1 29195 −20 221atttatcaacttccacc 2-11-4 ATttatcaacttcCACC 221_1 29906 −22 222cgtttaagaccaggcac 4-11-2 CGTTtaagaccaggcAC 222_1 30020 −22 223acaaaggaactcaggaagag 3-15-2 ACAaaggaactcaggaagAG 223_1 30424 −20 224tcacagacaagcaccaa 4-11-2 TCACagacaagcaccAA 224_1 31150 −20 225tactttttaaaacacgtagg 4-14-2 TACTttttaaaacacgtaGG 225_1 31329 −21 226gcacaatcacaaagaccaa 4-13-2 GCACaatcacaaagaccAA 226_1 31531 −22 227tcagtaaagaacagaggc 2-13-3 TCagtaaagaacagaGGC 227_1 31820 −20 228catatttccaccacacaag 2-15-2 CAtatttccaccacacaAG 228_1 32222 −21 229agtaaaccactgtcca 2-11-3 AGtaaaccactgtCCA 229_1 32601 −21 230tcctctttggcgatata 2-12-3 TCctctttggcgatATA 230_1 33337 −21 231cataaatacccctgaatac 2-14-3 CAtaaatacccctgaaTAC 231_1 33986 −20 232cgattttatcaccaaca 4-11-2 CGATtttatcaccaaCA 232_1 34175 −20 233acaatcaggttaagtgtgga 2-16-2 ACaatcaggttaagtgtgGA 233_1 34771 −21 234gaagccaaagactacca 3-12-2 GAAgccaaagactacCA 234_1 35096 −20 235tggtagggactgaattttaa 2-15-3 TGgtagggactgaatttTAA 235_1 35850 −21 236cggtagtcaatcacc 2-10-3 CGgtagtcaatcACC 236_1 36584 −20 237ctatcaaaattatttcacct 2-14-4 CTatcaaaattatttcACCT 237_1 36886 −22 238acgaaaatttagcatcctaa 3-14-3 ACG aaaatttagcatccTAA 238_1 37041 −20 239tggtaaacactgggc 2-11-2 TGgtaaacactggGC 239_1 38059 -19 240gattgttggttgtcatg 3-11-3 GATtgttggttgtcATG 240_1 38173 −21 241aattataccccacatttca 3-14-2 AATtataccccacatttCA 241_1 38806 −22 242tgcaattagacacgttacg 2-13-4 TGcaattagacacgtTACG 242_1 39004 −22 243ggcaaccaattaaacta 4-11-2 GGCAaccaattaaacTA 243_1 40226 −21 244cagttttatgctaatca 4-11-2 CAGTtttatgctaatCA 244_1 40272 −20 245gcaaaggaggagcggaataa 2-16-2 GCaaaggaggagcggaatAA 245_1 40707 −21 246aaacagaagtaagaaggtcc 2-14-4 AAacagaagtaagaagGTCC 246_1 41156 −22 247tccacttatccatagaaa 2-12-4 TCcacttatccataGAAA 247_1 41477 −20 248gctttgacgaacaggaaat 2-14-3 GCtttgacgaacaggaAAT 248_1 42282 −21 249ttccgaccaaaagaaaagac 4-14-2 TTCCgaccaaaagaaaagAC 249_1 42632 −21 250gcgacacgatccgttaaa 3-13-2 GCGacacgatccgttaAA 250_1 43104 −22 251ccccgttttaaaaac 4-9−2  CCCCgttttaaaaAC 251_1 43334 −20 Motif sequencesrepresent the contiguous sequence of nucleobases present in theoligonucleotide.

Designs refer to the gapmer design, F-G-F′, where each number representsthe number of consecutive modified nucleosides, e.g. 2′ modifiednucleosides (first number=5′ flank), followed by the number of DNAnucleosides (second number=gap region), followed by the number ofmodified nucleosides, e.g2′ modified nucleosides (third number=3′flank), optionally preceded by or followed by further repeated regionsof DNA and LNA, which are not necessarily part of the contiguoussequence that is complementary to the target nucleic acid.

Compound Chemistry

Each one compound from the two chemical series DHQ and THP weresynthesized to be suitable for the Y3H screening performed byHYBRIGENICS SERVICES SAS. Both compounds included PEG5 linker and weretagged with a Trimethoprim (TMP) anchor ligand (Table 5).

TABLE 5 TMP-tagged small molecule compound IDs Hybrigenics ID StructureDHQ compound- TMP HBX129653

THP compound- TMP HBX129654

Y3H ULTImate YChemH™ Screen

The two compounds were provided by Roche to HYBRIGENICS SERVICES SAS andtested for permeability and toxicity. Compounds were then screenedagainst HYBRIGENICS's cDNA Human placenta library (PLA). The screenswere carried out according to the optimized cell-to-cell mating protocoldeveloped for Hybrigenics ULTImate Y2H™ using at different compoundconcentration (Table 6).

TABLE 6 YChemH screens IDs Hybrigenics YChemH Probe ID Project YChemHscreen Project ID concentration DHQ HBX129653 hgx4240PLA_RP6_hgx4240v1_pB409_A  5 μM compound THP HBX129654 hgx4241PLA_RP6_hgx4241v1_pB409_A 10 μM compound

Y3H ULTImate YChemH™ Dependency Assay

Clones obtained from the screen were picked in 96-well format and clonespositive for growth under selective conditions (HIS+) were evaluated ina dependency assay using spot assays. Only clones that were able to growon selective medium in the presence of the tagged compound were beingpicked up, processed (cell lysis, PCR, gene sequencing) and mapped forprotein alignment using Blast analysis.

Y3H ULTImate YChemH™ 1-by-1 Validation Experiment—Prey Fragments

In this validation step each one identified fragment prey and onechemical probe (HBX129653, HBX129654) is tested in a 1-by-1 experiment.The plasmids from 3 selected preys from the screening library wereextracted from the yeast cells, amplified in E. coli and re-transformedinto YHGX13 yeast cells. For each interaction, DO1, 1/10, 1/100 and1/1000 of the diploid yeast culture expressing both hook and preyconstructs were spotted on a selective medium without tryptophan,leucine and histidine and supplemented with the chemical probe andFK506. Interactions were tested in duplicate. One plate was used perchemical compound and concentration (DMSO, 5, 10 and 20 μM of HBX129653,5, 10 and 20 μM of HBX129654, 5 μM of HBX24786 Trimethoprim (TMP) and 5μM of HBX129634 (TMP-PEGS-OH)). Plates were incubated at 30° C. for 3days. p Y3H ULTImate YChemH™ 1-by-1 Validation Experiment—Full LengthProteins

The coding sequence of full-length PAPD5var1 (NM_001040284.2) andPAPD7varX1 (XM_005248234.2) were reconstituted from an N-terminalcodon-optimized gene fragment (to remove high GC content) andcommercially available clones of the C-terminal regions of the proteinsand cloned in frame with the Gal4 Activation Domain (AD) into plasmidpP7 (AD-Prey), derived from the original pGADGH (Bartel et al., 1993 inCellular interactions in development: A practical approach. ed. Hartley,D. A., Oxford University Press, Oxford, pp. 153-179). The constructswere checked by sequencing the entire inserts. For each prey, amini-mating was carried out between YHGX13 (Y187ade2-101::loxP-kanMX-IoxP, mata) transformed with the prey plasmids andYPT6AT yeast cells (mata) transformed with the DHFR hook (Dihydrofolatereductase) to produce a diploid yeast culture. For each interaction,DO1, 1/10, 1/100 and 1/1000 of the diploid yeast culture expressing bothhook and prey constructs were spotted on a selective medium withouttryptophan, leucine and histidine and supplemented with the chemicalprobe and FK506. Interactions were tested in duplicate. One plate wasused per chemical compound and concentration (DMSO, 5, 10 and 20 μM ofHBX129653, 5, 10 and 20 μM of HBX129654, 5 μM of HBX24786 Trimethoprim(TMP) and 5 μM of HBX129634 (TMP-PEG5-OH)). Plates were incubated at 30°C. for 3 days.

Y3H ULTImate YChemH™—Competition with Free Compound

The competition assay is based on the previously described 1-by-1validation with a constant concentration for the chemical probe(HBX129653, HBX 129654) and increasing concentrations of the parentcompound of the chemical probe (MOL653, MOL654) or its inactiveenantiomer (INACT653, INACT654) (Table 7). The competition assays wereperformed on selective medium at 8 concentrations of the free compound(0, 0.25, 0.5, 1, 2, 5, 10 and 20 μM) and a consistent concentration forthe tagged Y3H-compound (1 μM).

TABLE 7 YChemH competition IDs Hybrigenics ID Structure DHQ compound-active MOL653

DHQ compound- inactive INACT653

THP compound- active MOL654

THP compound- inactive INACT654

HepaRG Cell Culture

HepaRG cells (Biopredics International, Rennes, France, Cat #HPR101)were cultured at 37° C. in a humidified atmosphere with 5% CO2 incomplete HepaRG growth medium consisting of William's E Medium (GIBCO),Growth Medium Supplement (Biopredics, Cat# ADD710) and 1% (v/v)GlutaMAX-I (Gibco #32551) and 1× Pen/Strep (Gibco, #15140) for 2 weeks.To initiate differentiation, 0.9% (v/v) DMSO (Sigma-Aldrich, D2650) wasadded to the growth medium on confluent cells. After one week, mediumwas replaced by complete differentiation medium (HepaRG growth mediumsupplemented with 1.8% (v/v) DMSO) in which cells were maintained forapproximately 4 weeks with differentiation medium renewal every 7 days.Differentiated HepaRG cells (dHepaRG), displayed hepatocyte-like cellislands surrounded by monolayer of biliary-like cells. Prior to HBVinfection and compound treatment, dHepaRG cells were seeded intocollagen I coated 96-well plates (Gibco, Cat# A11428-03) at 60,000 cellsper well in 100 μL of complete differentiation medium. Cells wereallowed to recover their differentiated phenotype in 96-well plates forapproximately 1 week after plating prior to HBV infection.

HBV Infection of dHepaRG Cells

dHepaRG cells were infected with HBV particles at an MOI of 30. The HBVparticles were produced from HBV-producing HepG2.2.15 cells (Sells et al1987 Proc Natl Acad Sci USA 84, 1005-1009). dHepaRG culture conditions,differentiation and HBV infection have been described previously (Hantz,2009, J. Gen. Virol., 2009, 90: 127-135). In brief completedifferentiation medium (HepaRG growth medium consisting of William's EMedium (GIBCO), Growth Medium Supplement (Biopredics, Cat #ADD710) and1% (v/v) GlutaMAX-I (Gibco #32551) and 1× Pen/Strep (Gibco, #15140),supplemented with 1.8% (v/v) DMSO), containing 4% PEG-8000 and virusstock (20 to 30 GE/cell) was added (120 μL/well). One daypost-infection, the cells were washed three times withphosphate-buffered saline and medium (complete differentiation medium)was replaced every two days during the experiment.

siRNA Treatment of HBV-Infected HepaRG

A pool of four different siRNAs was acquired from GE Dharmacon (ONTARGETplus) (Table 8).

TABLE 8  Overview siRNAs siRNA SEQ ON TARGETplus (Cat.No.)Target Sequence ID NO siPAPD5 J-010011-05 CAUCAAUGCUUUAUAUCGA 252(Cat. No. # J-010011-06 GGACGACACUUCAAUUAUU 253 L-010011-00- J-010011-07GAUAAAGGAUGGUGGUUCA 254 0010) J-010011-08 GAAUAGACCUGAGCCUUCA 255siPAPD7  J-009807-05 GGAGUGACGUUGAUUCAGA 256 (Cat. No. # J-009807-06CGGAGUUCAUCAAGAAUUA 257 L-009807-00- J-009807-07 CGGAGUUCAUCAAGAAUUA 2580005) J-009807-08 GCGAAUAGCCACAUGCAAU 259

One day before infection with HBV cells and 4 days after infection cellswere treated with the siRNA pool either against PAPD5, PAPD7, both orthe non-targeting siRNA as control. The siRNAs (25 nM each) weretransfected using DharmaFect 4 (GE Dharmacon; Cat. No. T-2004-01) andOPTI-MEM (Thermo Scientific; Cat. No. 51985034) according tomanufacturer's protocol. The experiment was run for 11 days.

HBV Antigen Measurements

To evaluate the impact on HBV antigen expression and secretion,supernatants were collected on Day 11. HBV HBsAg and HBeAg levels weremeasured using CLIA ELISA Kits (Autobio Diagnostic #CL0310-2,#CL0312-2), according to the manufacturer's protocol. Briefly, 25 μL ofsupernatant per well were transferred to the respective antibody coatedmicrotiter plate and 25 μL of enzyme conjugate reagent were added. Theplate was incubated for 60 min on a shaker at room temperature beforethe wells were washed five times with washing buffer using an automaticwasher. 25 μL of substrate A and B were added to each well. The plateswere incubated on a shaker for 10 min at room temperature beforeluminescence was measured using an Envision luminescence reader (PerkinElmer).

Cell Viability

After the removal of supernatant media from the HBV infected dHepaRGcells, cells were incubated with CellTiterGlo One Solution (Promega) tomeasure cell viability.

After LNA oligonucleotide treatments of HBV infected dHepaRG cells, cellviability was measured using the Cell Counting kit-8 (Sigma-Aldrich,#96992) according to the manufacturer's protocol. Briefly, Medium wasremoved from the cells and replaced by 110 μL of medium containing 9%Cell Counting kit-8. Cells were incubated for 1 h at 37° C. Thesupernatant was transferred into a new 96 wells plate and the absorbanceat 450 nm was measured using an Envision luminescence reader (PerkinElmer).

Real-Time PCR for Intracellular mRNA

For intracellular mRNA isolation, dHepaRG were washed once with PBS(Gibco) and lysed using the MagNA Pure “96 Cellular RNA Large VolumeKit” (Roche #05467535001). The lystates may be stored at at −80° C. Forthe real-time qPCR reaction an AB7900 HT sequence detection system(Applied Biosystems), the TaqMan® Gene Expression Master Mix(ThermoFisher Scientific) were used. For detection of HBV mRNA HBVcore-specific primer (Integrated DNA Technologies) (Table 9) and tomeasure reduction of PAPD5 and PAPD7, in the presence of siRNA,gene-specific TaqMan ® Expression Assay probes (ThermoFisher Scientific;PAPD5 Cat. No. 4331182; PAPD7 Cat. No. 4331182) were used. Samples werenormalized using TaqMan® Expression Assay probe against b-Actin(ThermoFisher Scientific; PAPD5 Cat. No. 4331182).

TABLE 9  HBV core specific TaqMan probes SEQ ID Name Dye Sequence NO HBVForward CTG TGC CTT GGG  265 core (F3_HBVcore) TGG CTT T Primer ReverseAAG GAA AGA AGT 266 (R3_HBVcore) CAG AAG GCAAAA Probe FAM-AGC TCC AAA/ZEN/ 267 (P3_HBVcore) MGB TTC TTT ATA AGG GTC GAT GTC CAT G

HBV DNA Extraction and Quantification from Virus Preparation

HBV DNA extraction is performed using the QIAamp UltraSens Virus kit(Qiagen, #53704) according to the manufacturer's protocol with thefollowing optimizations. 30 μL and 3 μL of the virus sample are dilutedinto 1 mL of PBS before adding buffer AC. The first centrifugation stepis done for 45 min at full speed and 4° C. HBV DNA is quantified induplicate by qPCR using a QuantStudio 12K Flex (Applied Biosystems), theTaqMan Gene Expression Master Mix (Applied Biosystems, #4369016) and apremix 1:1:0.5 of the primers indicated in Table 9 above and probereconstituted at 100 μM. The qPCR is performed using the followingsettings: UDG incubation (2 min, 50° C.), enzyme activation (10 min, 95°C.) and qPCR (40 cycles with 15 sec, 95° C. for denaturation and 1 min,60° C. for annealing and extension). Genomes equivalent calculation isbased on a standard curve generated from HBV genotype D plasmiddilutions with known concentrations.

Oligonucleotide Synthesis

Oligonucleotide synthesis is generally known in the art. Below is aprotocol which may be applied. The oligonucleotides of the presentinvention may have been produced by slightly varying methods in terms ofapparatus, support and concentrations used.

Oligonucleotides are synthesized on uridine universal supports using thephosphoramidite approach on an Oligomaker 48 at 1 μmol scale. At the endof the synthesis, the oligonucleotides are cleaved from the solidsupport using aqueous ammonia for 5-16hours at 60° C. Theoligonucleotides are purified by reverse phase HPLC (RP-HPLC) or bysolid phase extractions and characterized by UPLC, and the molecularmass is further confirmed by ESI-MS.

Elongation of the Oligonucleotide:

The coupling of β-cyanoethyl-phosphoramidites (DNA-A(Bz), DNA-G(ibu),DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), or LNA-T)is performed by using a solution of 0.1 M of the 5′-O-DMT-protectedamidite in acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile(0.25 M) as activator. For the final cycle, a phosphoramidite withdesired modifications can be used, e.g. a C6 linker for attaching aconjugate group or a conjugate group as such. Thiolation forintroduction of phosphorthioate linkages is carried out by usingxanthane hydride (0.01 M in acetonitrile/pyridine 9:1). Phosphordiesterlinkages can be introduced using 0.02 M iodine in THF/Pyridine/water7:2:1. The rest of the reagents are the ones typically used foroligonucleotide synthesis.

For post solid phase synthesis conjugation a commercially available C6aminolinker phorphoramidite can be used in the last cycle of the solidphase synthesis and after deprotection and cleavage from the solidsupport the aminolinked deprotected oligonucleotide is isolated. Theconjugates are introduced via activation of the functional group usingstandard synthesis methods.

Purification by RP-HPLC:

The crude compounds are purified by preparative RP-HPLC on a PhenomenexJupiter C18 10 μ 150×10 mm column. 0.1 M ammonium acetate pH 8 andacetonitrile is used as buffers at a flow rate of 5 mL/min. Thecollected fractions are lyophilized to give the purified compoundtypically as a white solid.

Abbreviations:

DCI: 4,5-Dicyanoimidazole

DCM: Dichloromethane

DMF: Dimethylformamide

DMT: 4,4′-Dimethoxytrityl

THF: Tetrahydrofurane

Bz: Benzoyl

Ibu: Isobutyryl

RP-HPLC: Reverse phase high performance liquid chromatography

T_(m) Assay:

Oligonucleotide and RNA target (phosphate linked, PO) duplexes arediluted to 3 mM in 500 ml RNase-free water and mixed with 500 ml 2×T_(m)-buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM Naphosphate, pH 7.0). Thesolution is heated to 95° C. for 3 min and then allowed to anneal inroom temperature for 30 min. The duplex melting temperatures (T_(m)) ismeasured on a Lambda 40 UV/VIS Spectrophotometer equipped with a Peltiertemperature programmer PTP6 using PE Templab software (Perkin Elmer).The temperature is ramped up from 20° C. to 95° C. and then down to 25°C., recording absorption at 260 nm. First derivative and the localmaximums of both the melting and annealing are used to assess the duplexT_(m).

EXAMPLE 1 DHQ and THP Binds to PAPD5 and PAPD7

PAPD5/7 were identified in Y3H Ultimate YChemH screen as commoninteraction partner of DHQ and THP

Both proteins PAPD5 (variant 1: NP_001035374; variant 2: NP_001035375)and PAPD7 (XP_005248291) were identified by a numerous number offragments in the Y3H screen for both compounds (DHQ and THP) asdescribed in the Materials and Method section. The identified proteinswere ranked with a confidence score of A (scale A-D) by HYBRIGENICS(Table 10).

TABLE 10 YChemH screen results for PAPD5/7 Hybrigenics Protein prey # ofConfidence ID identified fragments score DHQ HBX129653 PAPD5 variant 128 A compound PAPD5 variant 2 1 N/A PAPD7 12 A THP HBX129654 PAPD5variant 1 5 N/A compound PAPD5 variant 2 49 A PAPD7 24 A

PAPD5/7 Interaction with DHQ and THP Could be Confirmed using Y3HULTImate YChemH 1-by-1 Validation of Identified Prey Fragments andFurther with Full Length Proteins

In a first validation step three fragments identified in the firstscreen were selected for the 1-by-1 validation assay (as described inthe Materials and Method section) and tested at three differentconcentrations (5, 10 and 20 μM) (Table 11).

TABLE 11 interacting fragment selected for validation assay Interaction# Prey fragment ID Protein Prey A PLA_RP6_hgx4240v1_pB409_A-15 PAPD7 BPLA_RP6_hgx4241v1_pB409_A-112 PAPD5 variant 1 CPLA_RP6_hgx4240v1_pB409_A-24 PAPD5 variant 2

All three fragments could be validated as specific binders for DHQ andTHP already at the lowest tested concentration (FIG. 1 ).

In a second validation step, full length proteins for PAPD5 and PAPD7were synthesized and used for 1-by-1 validation (as described in theMaterials and Method section) with DHQ and THP (Table 12).

TABLE 12 Reference ID for full length protein prey used in 1-by-1validation assay Interaction # HYBRIGENICS Reference Protein Prey Ahgx4386v1_pP7 PAPD5 var1 full length B hgx4388v2_pP7 PAPD7 var1 fulllength

The interaction between these full length proteins and the DHQ and THPcompounds were confirmed at the lowest tested concentration and shown tobe specific for the chemical probes (FIG. 2 ).

PAPD5/7 Interaction with DHQ and THP in Y3H can be Competed by Both FreeActive Compound, but not the Inactive Enantiomer

After validation of binding of DHQ and THP to protein fragments and fulllength PAPD5 and PAPD7 the binding was confirmed in a Y3H ULTImateYChenH competition experiment (as described in the Materials and Methodsection) using either inactive or active free compound (Table 13). Adecrease of loss of yeast growth in the presence of the parent activecompound, but not in the presence of the inactive enantiomer, means thatthe parent compound competes with the chemical probe and interacts withthe protein target.

For all tested compounds toxicity on non-selective medium at the highestconcentration (20 μM) was tested using CellTiter-Glo Luminescent CellViability Assay (Promega) according to the manufacturer's protocol. Notoxicity was observed at this concentration for any compound as yeastgrowth was not affected (data not shown). For both active free parentcompounds (DHQ and THP, MOL653 and MOL654, respectively) competitioncould be observed, with lower concentration needed for competing thebinding to the full length protein than for the fragment interactions(FIG. 3 +4). Successful cross competition suggests a shared binding sidefor DHQ and THP to PAPD5/7 or at least binding in close proximity toeach other.

TABLE 13 Reference ID for protein prey used in competition assayInteraction # Prey fragment ID Protein Prey APLA_RP6_hgx4241v1_pB409_A-112 PAPD5 var1 experimental fragment Bhgx4386v1_pP7 PAPD5 var1 full length C PLA_RP6_hgx4240v1_pB409_A-15PAPD7 varX1 experimental fragment D hgx4388v2_pP7 PAPD7 varX1 fulllength

EXAMPLE 2 Inhibition of PAPD5 and/or PAPD7 with siRNA Results inEffective Treatment of HBV Infection

To correlate the binding of DHQ and THP to PAPD5/7 and the impact ofthese two proteins on HBV gene expression, we used RNAi technology toreduce these proteins in naturally HBV infected dHepaRG and to monitorthe impact of this reduction on viral parameters. For that we used siRNApools against PAPD5 and PAPD7 (see table 8) in HBV infected dHepaRGcells as described in the Materials and methods section.

Reduction of PAPD5 led to inhibition of viral expression measured bysecreted HBsAg and HBeAg as well as intracellular HBV mRNA (measuredusing CLIA ELISA and real-time PCR as described in the Materials andMethods section). While the reduction of PAPD5 mRNA dramatically reducedHBV gene expression, inhibition of PAPD7 had a modest effect on HBVexpression (FIG. 7 ). However, an enhanced synergistic anti-HBV activitywas observed when siRNA against PAPD7 and PAPD5 were combined (FIG. 7 ),suggesting a compensative role for PAPD7 in the absence of PAPD5.

EXAMPLE 3 DHQ and THP Effectively Reduces HBsAg and HBeAg

The potency of DHQ and THP and their variants against HBV infection weremeasured in HepG2.2.15 cells using HBsAg and HBeAg as read out.

HepG2.2.15 cells (Sells et al 1987 Proc Natl Acad Sci USA 84, 1005-1009)were cultured in 96 well plates (15.000 cells/well in 100 uL) inDMEM+GluTaMax-1 (GiBCO Cat. NO. 10569), 1% Pen Strep (Gibco Cat. No.15140), 10% FBS (Clontech Cat.No. 631106), Geneticin 0.25 ug/ml(Invitrogen 10131035). The compounds were tested using three-fold serialdilutions in DMSO with a top concentration of 100 μM and 9 serialdilutions. Each compound was tested in quadricate. The cells wereincubated for 3 days, supernatants were collected and HBsAg and HBeAgwere measured as described in the Materials and Methods section.

The IC₅₀ values of the tested compounds in the reduction of secretion ofHBsAg and HBeAg are shown in the following:

HBX129653 (DHQ-TMP): IC₅₀ HBsAg 1.181 uM

HBX129654 (THP-TMP): IC₅₀ HBsAg 0.299 uM

MOL653 (DHQ-free—active): IC₅₀ HBsAg 0.003 uM; IC₅₀ HBeAg 0.007 uM

MOL654 (THP-free—active): IC₅₀ HBsAg 0.003 uM

INACT653 (DHQ-free—inactive): IC₅₀ HBsAg 3.15 uM

INACT654 (THP-free—inactive): IC₅₀ HBsAg >25 uM

EXAMPLE 4 Screening for In Vitro Efficacy of Antisense OligonucleotidesTargeting PAPD5

An oligonucleotide screen was done across the PAPD5 mRNA using 16 to 20mer gapmers. Efficacy testing was performed in an in vitro experiment inHeLa cells.

Cell Lines

HeLa cell line was purchased from European Collection of AuthenticatedCell Cultures (ECACC, #93021013) and maintained as recommended by thesupplier in a humidified incubator at 37° C. with 5% CO2. For assays,2,500 cells/well were seeded in a 96 multi well plate in Eagle's MinimumEssential Medium (Sigma, M2279) with 10% fetal bovine serum (FBS), 2 mMGlutamin AQ, 1% NEAA, 25 μg/ml Gentamicin.

Oligonucleotide Efficacy

Cells were incubated for 24 hours before addition of oligonucleotidesdissolved in PBS. Final concentration of oligonucleotides was 5 and 25μM, the final culture volume was 100 μl/well. The cells were harvested 3days after addition of oligonucleotide compounds and RNA was extractedusing the PureLink Pro 96 RNA Purification kit (Ambion), according tothe manufacturer's instructions. After RNA/LNA duplex denaturation (90°C., 40 sec) QPCR was done with a one-step protocol (gScript™ XLTOne-Step RT-qPCR ToughMix®, Low ROX™ from Quanta Bioscience, #95134-500)in a duplex set up with TaqMan primer assays for the gene of interestPAPD5 (Hs00223727_m1, FAM-MGB, Life Technologies) and a house keepinggene GUSB (Hu_4326320E, VIC-MGB, Life Technologies) following therecommendations of the provider. The relative PAPD5 mRNA expressionlevel is shown in table 14 as % of average control samples (PBS-treatedcells). FIG. 8 presents the target knockdown achieved with theindividual oligonucleotide compounds across the PAPAD5 encodingsequence.

TABLE 14  in vitro efficacy of anti-PAPD5 compounds(single experiment with duplex QPCR).PAPD5 mRNA levels are normalized to GUSB in HeLa cellsand shown as % of control (PBS treated cells). CMP % mRNA of Start on IDConcentration control SEQ ID NO μM Avg sd Compound (CMP) NO 10 12_1 2570.8 9.3 AGggtagatgtgtttaACT 1656 12_1 5 59.3 11.7 AGggtagatgtgtttaACT1656 13_1 25 43.9 0.2 CAGcctaaacttagtGG 2000 13_1 5 53.2 18.1CAGcctaaacttagtGG 2000 14_1 25 43.9 8.8 AAgccctcaatgtaaaACAC 2446 14_1 596.6 8.1 AAgccctcaatgtaaaACAC 2446 15_1 25 21.1 3.2 AATagcaagtagaggaGAG3059 15_1 5 52.9 17.1 AATagcaagtagaggaGAG 3059 16_1 25 28.5 7.3AAataaggatactgGCGA 4036 16_1 5 68.6 16.4 AAataaggatactgGCGA 4036 17_1 2550.2 20.4 GAGGgaacacataataaaAG 4484 17_1 5 72.9 29.0GAGGgaacacataataaaAG 4484 18_1 25 19.7 4.0 GTaatacctctcacATTC 5928 18_15 53.8 14.0 GTaatacctctcacATTC 5928 19_1 25 37.0 8.4 AGtaacaccaatctcATTG6652 19_1 5 58.8 10.7 AGtaacaccaatctcATTG 6652 20_1 25 66.1 5.3GTgacagtattcaatGATC 7330 20_1 5 58.4 12.5 GTgacagtattcaatGATC 7330 21_125 33.2 3.1 CAgttccgtatcaccAAC 7702 21_1 5 45.3 11.6 CAgttccgtatcaccAAC7702 22_1 25 56.3 6.7 AAgtctaactcaaagccATC 8292 22_1 5 70.1 16.8AAgtctaactcaaagccATC 8292 23_1 25 21.7 nd AGgcttccattttattGAA 8625 23_15 44.3 0.5 AGgcttccattttattGAA 8625 24_1 25 71.2 10.2 TTttagaaaacgagGCTA9866 24_1 5 63.8 10.6 TTttagaaaacgagGCTA 9866 25_1 25 13.4 0.1GTAttcttattcttgCT 10254 25_1 5 27.9 10.6 GTAttcttattcttgCT 10254 26_1 2512.0 0.9 ATTAttcccacagtaaGA 10881 26_1 5 37.7 1.1 ATTAttcccacagtaaGA10881 27_1 25 57.5 4.4 AACaacaaacaggatggGC 11370 27_1 5 110.0 2.2AACaacaaacaggatggGC 11370 28_1 25 35.2 0.8 ATATccacaatattctgAT 1179028_1 5 47.7 1.2 ATATccacaatattctgAT 11790 29_1 25 77.8 32.7AAagaaataatgtcgtCTGG 12413 29_1 5 73.9 2.5 AAagaaataatgtcgtCTGG 1241330_1 25 23.4 4.0 CCAgagtaaacaaaTCC 12718 30_1 5 48.0 2.0CCAgagtaaacaaaTCC 12718 31_1 25 57.7 10.6 ATtcaacatttttagtcACC 1355531_1 5 53.6 4.8 ATtcaacatttttagtcACC 13555 32_1 25 32.8 5.7TTTggtaattctttlltTAG 14297 32_1 5 33.1 5.4 TTTggtaattctttlltTAG 1429733_1 25 58.0 nd CAatgaggaaacaagaGTCA 15137 33_1 5 80.9 17.9CAatgaggaaacaagaGTCA 15137 34_1 25 56.3 9.0 TTCaaaataatgtgggaGGT 1569534_1 5 63.6 2.7 TTCaaaataatgtgggaGGT 15695 35_1 25 26.4 1.9GGatatttgatacggcaAAT 16145 35_1 5 39.1 10.1 GGatatttgatacggcaAAT 1614536_1 25 77.5 12.6 CTAtaagaagcaaaCCC 16714 36_1 5 76.6 2.7CTAtaagaagcaaaCCC 16714 37_1 25 45.3 4.5 ATAtaattcacgtttcaCTT 17097 37_15 91.9 nd ATAtaattcacgtttcaCTT 17097 38_1 25 28.9 2.7AATgattcacatgaaggTTA 17420 38_1 5 38.2 2.2 AATgattcacatgaaggTTA 1742039_1 25 18.0 4.7 GTtaggattttgcTATG 18299 39_1 5 29.2 4.1GTtaggattttgcTATG 18299 40_1 25 80.4 2.0 GTAcaaatatcaaccgTAT 18669 40_15 118.8 10.4 GTAcaaatatcaaccgTAT 18669 41_1 25 32.8 7.7CACactatttcaagatgcTA 19681 41_1 5 85.6 4.8 CACactatttcaagatgcTA 1968142_1 25 65.6 20.2 CACctatacaatggagtATT 20352 43_1 25 64.9 12.3ATcatacgtcattagaGAAC 20721 43_1 5 79.4 25.2 ATcatacgtcattagaGAAC 2072144_1 25 70.7 2.6 CAgaacagatactttgcCA 21111 44_1 5 84.7 5.5CAgaacagatactttgcCA 21111 45_1 25 49.8 11.9 AAgaatggttggttaaGGG 2178245_1 5 79.2 4.3 AAgaatggttggttaaGGG 21782 46_1 25 35.1 0.0AGAAttggtaaactggacTG 22378 46_1 5 55.1 7.7 AGAAttggtaaactggacTG 2237847_1 25 28.4 3.9 AGAattatattggcTGG 23160 47_1 5 49.6 2.2AGAattatattggcTGG 23160 48_1 25 60.8 13.7 CCtaaaccagacagaaaAGA 2399348_1 5 69.1 5.0 CCtaaaccagacagaaaAGA 23993 49_1 25 31.9 4.7ACCAattagagcagaaaTC 24813 49_1 5 62.2 1.0 ACCAattagagcagaaaTC 24813 50_125 41.3 6.5 TTCtaaataacagatggGTC 25047 50_1 5 64.4 21.7TTCtaaataacagatggGTC 25047 51_1 25 27.1 5.9 TTTataatttttttccaTCT 2608051_1  5 49.1 7.7 TTTataatttttttccaTCT 26080 52_1 25 34.9 6.8GCAAatatcagattaaccTC 26625 52_1 5 60.5 3.1 GCAAatatcagattaaccTC 2662553_1 25 25.9 0.8 AACggtatggcagaagaCAA 26973 53_1 5 43.6 2.2AACggtatggcagaagaCAA 26973 54_1 25 5.1 0.1 TTCAacctttactgcAT 27813 54_15 25.7 3.1 TTCAacctttactgcAT 27813 55_1 25 40.8 0.9 ACtgataaagggcattTCAA28357 55_1 5 64.5 1.6 ACtgataaagggcattTCAA 28357 56_1 25 6.4 2.3CAgtaggaatgtggCTT 28718 56_1 5 19.6 0.7 CAgtaggaatgtggCTT 28718 57_1 2523.9 0.4 TTTtatggcagggtttcAC 29327 57_1 5 41.2 7.9 TTTtatggcagggtttcAC29327 58_1 25 19.6 0.7 TCactgttaaaccTCAC 29902 58_1 5 59.0 12.9TCactgttaaaccTCAC 29902 59_1 25 53.1 4.8 CAATtttctaattcaatgGT 30704 59_15 72.4 29.8 CAATtttctaattcaatgGT 30704 60_1 25 42.8 2.0AAGAtataattcacccaCT 31008 60_1 5 77.3 13.7 AAGAtataattcacccaCT 3100861_1 25 47.4 2.7 GCcacataaaggataAAGT 31348 61_1 5 87.8 12.8GCcacataaaggataAAGT 31348 62_1 25 49.8 2.7 CCcattagaagtaaggtGA 3236762_1 5 80.1 11.1 CCcattagaagtaaggtGA 32367 63_1 25 49.4 1.1ATgtaaattaaaactTCCC 32632 63_1 5 67.2 1.7 ATgtaaattaaaactTCCC 32632 64_125 41.7 1.7 TGAgagcataaaagtacgGA 32945 64_1 5 73.1 6.3TGAgagcataaaagtacgGA 32945 65_1 25 19.4 0.2 TTCAcaacaggtaaagGG 3359365_1 5 47.7 3.3 TTCAcaacaggtaaagGG 33593 66_1 25 46.6 0.2TGcattcctaagtaacATAA 34801 66_1 5 52.0 5.3 TGcattcctaagtaacATAA 3480167_1 25 34.1 10.4 AGAgaaaagtgatgaggGAA 35368 67_1 5 45.5 2.0AGAgaaaagtgatgaggGAA 35368 68_1 25 51.7 3.6 ATAcggatcaccagctaAA 3613168_1 5 74.2 18.8 ATAcggatcaccagctaAA 36131 69_1 25 26.5 3.5CAtgttatgcacagaAGAT 36712 69_1 5 51.0 1.4 CAtgttatgcacagaAGAT 36712 70_125 68.8 11.0 CGctgaagaactaagtATTA 37282 70_1 5 75.2 6.7CGctgaagaactaagtATTA 37282 71_1 25 36.5 5.1 CAAacagatggtggtgaTA 3787071_1 5 66.3 6.4 CAAacagatggtggtgaTA 37870 72_1 25 62.8 9.4AGTagccattaggaTG 38478 72_1 5 80.2 3.1 AGTagccattaggaTG 38478 73_1 2559.9 11.1 ATacacaggctccataATA 39639 73_1 5 70.6 9.5 ATacacaggctccataATA39639 74_1 25 34.5 9.7 GATUttgtatagtccacAA 40178 74_1 5 43.3 6.9GATUttgtatagtccacAA 40178 75_1 25 59.8 0.2 GCatctataaaaaaggGACA 4104275_1 5 68.1 2.9 GCatctataaaaaaggGACA 41042 76_1 25 50.0 1.8AGtgcaagtatcGCT 41734 76_1 5 73.2 0.1 AGtgcaagtatcGCT 41734 77_1 25 45.83.2 CCAaaagaatcaagttcgTA 42442 77_1 5 75.0 14.2 CCAaaagaatcaagttcgTA42442 78_1 25 56.1 4.5 CCtcagaccaaatttATTT 43203 78_1 5 85.1 1.8CCtcagaccaaatttATTT 43203 79_1 25 27.1 3.4 TTCaacaagcatctattGTA 4366379_1 5 43.3 1.1 TTCaacaagcatctattGTA 43663 80_1 25 4.4 1.8CAAaggttgttgtacTCT 44220 80_1 5 19.2 3.4 CAAaggttgttgtacTCT 44220 81_125 35.9 11.3 TCAtaaatctttttccaCG 44756 81_1 5 67.6 0.7TCAtaaatctttttccaCG 44756 82_1 25 36.9 2.1 CTtgttacggatttaatGTG 4504282_1 5 67.5 10.5 CTtgttacggatttaatGTG 45042 83_1 25 59.1 8.2GCTataaaaatagaaGCC 46202 84_1 25 61.3 3.4 TCCttagcaaactaaaCAT 47142 84_15 92.6 0.3 TCCttagcaaactaaaCAT 47142 85_1 25 51.0 1.9AGCaaaaggcaggtattcAA 47843 85_1 5 92.2 5.5 AGCaaaaggcaggtattcAA 4784386_1 25 46.6 2.8 GAAtccatttacatattCAC 48267 86_1 5 79.2 0.9GAAtccatttacatattCAC 48267 87_1 25 49.7 0.0 TCcagtatccaaaacATAC 4925687_1  5 93.1 2.8 TCcagtatccaaaacATAC 49256 88_1 25 63.4 7.0AGCTtaaagaagaacggTT 49688 88_1 5 24.2 15.8 AGCTtaaagaagaacggTT 4968889_1 25 33.3 12.3 CAcaacgtgcctaccTT 50508 89_1 5 71.9 32.7CAcaacgtgcctaccTT 50508 90_1 25 80.3 18.2 CCAgaatccaagaaaatGG 50764 90_15 112.9 36.7 CCAgaatccaagaaaatGG 50764 91_1 25 88.9 10.3TCacctcgaactaaacaaGT 51561 91_1 5 90.1 5.9 TCacctcgaactaaacaaGT 5156192_1 5 26.4 7.9 GTatctttctgtacTATT 52461 93_1 25 15.1 7.2GTCAttctactaacaaaCG 53305 93_1 5 29.7 15.4 GTCAttctactaacaaaCG 5330594_1 25 48.1 20.2 TGgaaaaggaagaacCATT 53865 94_1 5 42.8 34.7TGgaaaaggaagaacCATT 53865 95_1 25 59.2 3.6 AAtacaactcttccgTGAT 5463895_1 5 73.0 9.2 AAtacaactcttccgTGAT 54638 96_1 25 36.8 ndAAtaccctgacgagCTG 54942 96_1 5 75.2 4.1 AAtaccctgacgagCTG 54942 97_1 2540.0 20.5 TCAtaaaacatgatccttGC 55741 97_1 5 96.4 3.0TCAtaaaacatgatccttGC 55741 98_1 25 55.7 1.0 CTAaagcagatccataGAA 5627798_1 5 103.3 24.2 CTAaagcagatccataGAA 56277 99_1 25 34.5 16.3AGActataacttttgctaCA 56942 99_1 5 73.1 6.0 AGActataacttttgctaCA 56942100_1  25 34.1 nd AGcaatgacttgaacataGT 57369 100_1  5 110.9 22.8AGcaatgacttgaacataGT 57369 101_1  25 87.5 16.7 ATAaaacaagcatacggGC 58146101_1  5 98.8 15.0 ATAaaacaagcatacggGC 58146 102_1  25 47.2 11.2ATgagataccagcagatAG 58588 102_1  5 94.0 8.9 ATgagataccagcagatAG 58588103_1  25 52.0 4.5 AGAAgaaatcctgagtaaTC 59089 103_1  5 72.7 0.6AGAAgaaatcctgagtaaTC 59089 104_1  25 67.0 9.6 CCCtaaaaagtgacgTA 59461104_1  5 106.7 9.6 CCCtaaaaagtgacgTA 59461 105_1  25 35.8 4.4TTAAgttagatcacgGC 59970 105_1  5 54.6 9.3 TTAAgttagatcacgGC 59970 106_1 25 74.8 3.5 GTggatacagaaagCCA 60738 106_1  5 92.6 3.2 GTggatacagaaagCCA60738 107_1  25 92.2 6.9 GTAtcggcaggagATT 61038 107_1  5 121.5 31.7GTAtcggcaggagATT 61038 108_1  25 50.2 10.6 TAACtaattgattccattGC 61868108_1  5 66.4 4.6 TAACtaattgattccattGC 61868 109_1  25 43.3 8.4AGAagaacggaaattgCC 62418 109_1  5 72.9 4.1 AGAagaacggaaattgCC 62418110_1  25 59.3 0.2 ATAAtgattttcctatCC 62822 110_1  5 76.0 3.6ATAAtgattttcctatCC 62822 111_1  25 28.0 0.4 ATggttttgtggagaAGG 63000111_1  5 11.3 10.2 ATggttttgtggagaAGG 63000 112_1  25 97.4 4.2TGctgctgtgaaaagaAATG 63697 112_1  5 139.8 0.6 TGctgctgtgaaaagaAATG 63697113_1  25 83.7 7.7 GTgtccaattttttatTAT 64377 113_1  5 86.7 0.4GTgtccaattttttatTAT 64377 114_1  25 78.7 8.5 GAtggaatcaactgtgtaGT 65307114_1  5 38.6 17.3 GAtggaatcaactgtgtaGT 65307 115_1  25 31.4 3.6GATGgtgacaaattattCT 65894 115_1  5 19.6 4.4 GATGgtgacaaattattCT 65894116_1  5 21.7 nd TGcttttgggaatCTTT 66650 117_1  25 40.8 18.6GAtgtcctacaatgaacACG 68024 117_1  5 39.4 10.3 GAtgtcctacaatgaacACG 68024118_1  25 74.2 11.3 GTAcaaggacaaagtaACC 68732 118_1  5 35.4 7.7GTAcaaggacaaagtaACC 68732 119_1  25 102.8 9.8 TGAAacgcctatctcTA 69029119_1  5 96.9 2.6 TGAAacgcctatctcTA 69029 120_1  25 56.0 2.4ATActatttatgcttatgGA 69796 120_1  5 79.7 2.5 ATActatttatgcttatgGA 69796121_1  25 27.1 0.9 TTGtaatcaaggcaataAGG 70770 121_1  5 56.8 3.8TTGtaatcaaggcaataAGG 70770 122_1  25 24.4 1.4 GAAGtccaataacgcaGA 71091122_1  5 63.3 14.8 GAAGtccaataacgcaGA 71091 123_1  25 68.6 11.3ATAtccaatctctatatgTG 72013 123_1  5 85.3 6.3 ATAtccaatctctatatgTG 72013124_1  25 83.9 0.8 GCcttacacaagactatATT 73444 124_1  5 77.0 43.8GCcttacacaagactatATT 73444 125_1  25 93.0 7.4 TGCTgaattttatgttaAC 73823125_1  5 87.9 4.7 TGCTgaattttatgttaAC 73823 126_1  25 53.9 8.5CAcaagatgatgggttTAAG 74559 126_1  5 77.5 2.5 CAcaagatgatgggttTAAG 74559127_1  25 34.7 10.4 TTagtggtttgggtGC 75043 127_1  5 57.6 9.4TTagtggtttgggtGC 75043 128_1  25 71.7 nd AGattgttaccttactGAT 76110128_1  5 78.9 0.6 AGattgttaccttactGAT 76110 129_1  25 79.0 8.9TATtacaaatatcaatcTCC 76931 129_1  5 97.7 nd TATtacaaatatcaatcTCC 76931130_1  25 61.9 1.5 TACcaaaagcatagagtGG 77605 130_1  5 84.6 1.9TACcaaaagcatagagtGG 77605 131_1  25 50.3 1.8 AAttatcttcccgCTAC 78652131_1  5 64.9 13.4 AAttatcttcccgCTAC 78652

EXAMPLE 5 Screening for In Vitro Efficacy of Antisense OligonucleotidesTargeting PAPD7

The oligonucleotide screen across the PAPD7 mRNA was conductedessentially as described in Example 4, with the substitution of theTaqMan primer assays for the gene of interest PAPD7 (Hs00173159_m1,FAM-MGB, Life Technologies).

The relative PAPD7 mRNA expression level is shown in table 15 as % ofaverage control samples (PBS-treated cells). FIG. 9 presents the targetknockdown achieved with the individual oligonucleotide compounds acrossthe PAPAD5 encoding sequence.

TABLE 15  in vitro efficacy of anti-PAPD7 compounds(single experiment with duplex QPCR).PAPD5 mRNA levels are normalized to GUSB in HeLa cellsand shown as % of control (PBS treated cells). CMP Concentration% mRNA of control Start on ID NO μM Avg sd Compound (CMP) SEQ ID NO 10132_1 25 72.8 4.0 GTtgggtggaataggCA 132 132_1 5 79.1 0.3GTtgggtggaataggCA 132 133_1 25 25.6 1.4 TCgatttcccgttccAA 1962 133_1 542.7 0.7 TCgatttcccgttccAA 1962 134_1 25 65.2 2.3 ACAacctacacataaattGC2510 134_1 5 93.0 1.1 ACAacctacacataaattGC 2510 135_1 25 66.7 0.6ACtataagaactcccAACA 2668 135_1 5 58.9 13.0 ACtataagaactcccAACA 2668136_1 25 76.6 5.0 GAGAaaaagagttacaaGC 2695 136_1 5 76.8 16.6GAGAaaaagagttacaaGC 2695 137_1 25 53.7 12.7 AACTggagggagagaagAG 2883137_1 5 87.5 20.1 AACTggagggagagaagAG 2883 138_1 25 76.5 2.2TTtctaagagcagaggtACA 3090 138_1 5 102.6 44.0 TTtctaagagcagaggtACA 3090139_1 25 19.6 0.2 AGAAgtaacaagagcCT 3463 139_1 5 44.6 20.4AGAAgtaacaagagcCT 3463 140_1 25 54.6 3.0 AGtatcaaaccagacCTC 3795 140_1 539.9 9.9 AGtatcaaaccagacCTC 3795 141_1 25 32.6 5.1 CCACaaccgaaagacTT4205 141_1 5 51.2 1.9 CCACaaccgaaagacTT 4205 142_1 25 37.2 3.1AAtacacactgcattTTCA 4336 142_1 5 53.1 1.7 AAtacacactgcattTTCA 4336 143_125 26.2 6.0 CCaggtagatagcacAG 4686 143_1 5 80.5 4.2 CCaggtagatagcacAG4686 144_1 25 64.2 4.8 CCatgacaaagtaacaACAG 4821 144_1 5 66.4 0.7CCatgacaaagtaacaACAG 4821 145_1 25 37.4 3.1 CAgaatttcctttgagTTA 5134145_1 5 44.8 4.7 CAgaatttcctttgagTTA 5134 146_1 25 85.5 4.1CCttcgcaagaaagaattGA 5263 146_1 5 102.4 4.7 CCttcgcaagaaagaattGA 5263147_1 25 23.5 2.3 TCatacatacacgcttCT 5577 147_1 5 55.8 6.5TCatacatacacgcttCT 5577 148_1 25 30.6 5.8 TGcgaaaagattgGAGG 5945 148_1 532.4 5.1 TGcgaaaagattgGAGG 5945 149_1 25 72.9 11.4 CAcaggacgcttacatgaAT6235 149_1 5 93.9 15.5 CAcaggacgcttacatgaAT 6235 150_1 25 36.1 2.0GCTgtttttttttcttaAC 6352 150_1 5 54.3 10.9 GCTgtttttttttcttaAC 6352151_1 25 39.5 2.5 ACcataagtgagtgttCTT 6834 151_1 5 120.0 8.9ACcataagtgagtgttCTT 6834 152_1 25 90.6 9.7 ACacaagcccatagaaacAG 7158152_1 5 68.2 7.9 ACacaagcccatagaaacAG 7158 153_1 25 3.4 0.2CAgtagtaaccacCAAG 7447 153_1 5 21.6 4.8 CAgtagtaaccacCAAG 7447 154_1 2546.6 0.7 CCtgcaaacttttattTAT 7708 154_1 5 64.7 3.1 CCtgcaaacttttattTAT7708 155_1 25 15.7 1.3 ACttagtaatagcaGCA 8074 155_1 5 37.6 2.1ACttagtaatagcaGCA 8074 156_1 25 24.7 0.3 ATgaatactccgaagACTT 8249 156_15 59.8 3.1 ATgaatactccgaagACTT 8249 157_1 25 42.7 5.2AAAgaaaaggatcacaaGCC 8784 157_1 5 60.5 3.2 AAAgaaaaggatcacaaGCC 8784158_1 25 91.4 19.9 AGAcagaaatcacctaaCA 8887 158_1 5 82.3 4.9AGAcagaaatcacctaaCA 8887 159_1 25 19.2 2.4 TAGaacagacattattcATC 9506159_1 5 47.4 10.0 TAGaacagacattattcATC 9506 160_1 25 33.0 5.2AGttacacggagcagcAC 9664 160_1 5 57.4 3.6 AGttacacggagcagcAC 9664 161_125 82.6 22.1 CACtatacacagaacacTAT 9770 161_1 5 85.0 7.0CACtatacacagaacacTAT 9770 162_1 25 70.4 2.3 AGctgtctaaatacATGG 10000162_1 5 91.7 16.8 AG ctgtctaaatacATGG 10000 163_1 25 26.7 0.6ATGaacctattttatgcTTC 10206 163_1 5 31.9 1.8 ATGaacctattttatgcTTC 10206164_1 25 36.5 5.5 ACcatcattaacctgcGT 10318 164_1 5 50.0 0.5ACcatcattaacctgcGT 10318 165_1 25 129.2 5.9 AGtaaagtgcccagatGT 10568166_1 25 45.8 3.3 TTCcctatgaaatcctcAA 10781 166_1 5 87.1 14.5TTCcctatgaaatcctcAA 10781 167_1 25 35.6 2.8 CActcttcatagaatgCAAC 10917167_1 5 55.6 9.1 CActcttcatagaatgCAAC 10917 168_1 25 19.4 4.9AAtgcttaatttttctCTCT 11084 168_1 5 38.2 1.9 AAtgcttaatttttctCTCT 11084169_1 25 26.8 4.1 TTAgagacgatgcctatAAC 11308 169_1 5 57.6 2.5TTAgagacgatgcctatAAC 11308 170_1 25 26.1 2.2 TGAAtagttcccatagaTT 11585170_1 5 54.7 1.8 TGAAtagttcccatagaTT 11585 171_1 25 10.8 0.6CAGcataattgttttcTTT 12330 171_1 5 33.1 1.3 CAGcataattgttttcTTT 12330172_1 25 13.8 2.2 ATGTcattatgllttagTT 12634 172_1 5 33.8 3.1ATGTcattatgllttagTT 12634 173_1 25 22.6 2.5 CAgcagtatctcttaGAA 12902173_1 5 36.8 1.8 CAgcagtatctcttaGAA 12902 174_1 25 9.7 0.4CGgtaagggttcggTG 13126 174_1 5 18.2 0.0 CGgtaagggttcggTG 13126 175_1 2551.9 3.5 CATGaaccacattaggaAC 13383 175_1 5 67.8 15.0 CATGaaccacattaggaAC13383 176_1 25 54.6 0.0 CAttcaacacacacgACAA 13578 176_1 5 83.5 0.3CAttcaacacacacgACAA 13578 177_1 25 38.9 1.1 AAgtatccaagactcAAGA 13889177_1 5 47.6 3.4 AAgtatccaagactcAAGA 13889 178_1 25 49.8 9.4CCACagaaacaccgAG 14100 178_1 5 74.0 21.3 CCACagaaacaccgAG 14100 179_1 2569.6 0.2 TGGaaaagggaagggaaGA 14179 179_1 5 137.8 15.7TGGaaaagggaagggaaGA 14179 180_1 25 54.5 0.1 AGagagtccgaagccTG 14432180_1 5 77.7 18.8 AGagagtccgaagccTG 14432 181_1 25 27.6 5.5ATGggaaaggtaacgaGC 14616 181_1 5 79.5 nd ATGggaaaggtaacgaGC 14616 182_125 63.3 1.9 CTAtcctacaagtccgAA 15471 182_1 5 106.6 12.5CTAtcctacaagtccgAA 15471 183_1 25 9.2 0.6 CATTgcttttataatccTA 15816183_1 5 30.6 3.1 CATTgcttttataatccTA 15816 184_1 25 9.7 1.0CTTTttaaggacaggaGG 15988 184_1 5 40.9 0.7 CTTTttaaggacaggaGG 15988 185_125 79.7 0.2 GAtgaaagataagtgaGCAT 16395 185_1 5 76.9 2.5GAtgaaagataagtgaGCAT 16395 186_1 25 50.5 17.3 GAagcctgtaataattAAGC 17007186_1 5 54.4 10.9 GAagcctgtaataattAAGC 17007 187_1 25 95.4 6.2CACCctagtaaagcaaAC 17151 187_1 5 137.3 37.9 CACCctagtaaagcaaAC 17151188_1 25 15.8 2.5 GCAaatgtaagcctttTT 17303 188_1 5 25.3 0.4GCAaatgtaagcctttTT 17303 189_1 25 45.4 2.3 ACctgacagctaccgAC 17498 189_15 46.8 12.3 ACctgacagctaccgAC 17498 190_1 25 9.3 1.5 AAgagtgggttgtaAGC17963 190_1 5 19.1 2.0 AAgagtgggttgtaAGC 17963 191_1 25 15.3 2.6TAgtgaaaatatttggAGTT 18101 191_1 5 33.2 4.5 TAgtgaaaatatttggAGTT 18101192_1 25 62.7 7.6 TTtcagcaccttaaacCC 18518 192_1 5 84.9 12.6TTtcagcaccttaaacCC 18518 193_1 25 86.0 24.0 TTAAgggaaaggaaacgtCA 18747193_1 5 79.5 6.2 TTAAgggaaaggaaacgtCA 18747 194_1 25 14.4 1.0GTAggtaaagggcaaaggAA 19007 194_1 5 37.1 4.6 GTAggtaaagggcaaaggAA 19007195_1 25 13.4 0.5 GTgaattaaagccaAAGC 19252 195_1 5 51.9 9.1GTgaattaaagccaAAGC 19252 196_1 25 80.3 21.3 TGTUttgtattttagTAT 19476196_1 5 77.5 23.0 TGTUttgtattttagTAT 19476 197_1 25 24.2 1.1GAggtttlltttagtgAATT 19722 197_1 5 29.0 2.4 GAggtttlltttagtgAATT 19722198_1 25 20.6 1.7 GAGgagctaaacggaCA 20062 198_1 5 33.4 6.4GAGgagctaaacggaCA 20062 199_1 25 37.7 4.0 GTttagtcttatgttctcAC 20623199_1 5 38.8 5.6 GTttagtcttatgttctcAC 20623 200_1 25 23.8 2.1CAaatactgaatatgcCCG 20726 200_1 5 52.0 10.0 CAaatactgaatatgcCCG 20726201_1 25 13.6 0.4 ACCatttaaatcgccAAC 20926 201_1 5 54.0 8.8ACCatttaaatcgccAAC 20926 202_1 25 53.5 2.6 CAgtaagagtagcccaacAA 21234202_1 5 82.9 0.9 CAgtaagagtagcccaacAA 21234 203_1 25 31.9 8.8TAACggcaacatcaaataGC 22017 203_1 5 56.3 4.2 TAACggcaacatcaaataGC 22017204_1 25 35.9 5.8 ACtgagcaccaactaCAC 22592 204_1 5 67.9 10.4ACtgagcaccaactaCAC 22592 205_1 25 36.8 1.7 CCtaattttatgtatcACAT 23074205_1 5 96.2 8.4 CCtaattttatgtatcACAT 23074 206_1 25 97.7 1.3CTAGgaattataacaaatCA 23462 23509 207_1 25 88.0 17.6 ACtccaaagaacatacTCAC24628 207_1 5 97.0 4.8 ACtccaaagaacatacTCAC 24628 208_1 25 77.3 18.8TAAgagaaacaatcacaCCA 24935 208_1 5 124.2 2.5 TAAgagaaacaatcacaCCA 24935209_1 25 68.6 6.7 TGtcctaaaatatcagCAG 26022 209_1 5 102.4 4.6TGtcctaaaatatcagCAG 26022 210_1 25 54.8 0.4 CTtttaatgagacagtgCA 26212210_1 5 73.6 1.8 CTtttaatgagacagtgCA 26212 211_1 25 16.7 1.1TTGTagcataagatggaaAG 26500 211_1 5 47.0 1.3 TTGTagcataagatggaaAG 26500212_1 25 41.0 0.2 AAActgtagccaataacTGT 26945 212_1 5 92.7 9.4AAActgtagccaataacTGT 26945 213_1 25 68.3 1.6 ATtcatcctaacacaagTAG 27117213_1 5 74.6 6.7 ATtcatcctaacacaagTAG 27117 214_1 25 54.5 0.3CACGaaaggaacagctaAG 27264 214_1 5 82.0 13.5 CACGaaaggaacagctaAG 27264215_1 25 32.4 1.7 ACAAcaggcaagtaCC 27411 215_1 5 93.5 2.7ACAAcaggcaagtaCC 27411 216_1 25 40.0 2.5 GCTaaacactataagGAT 27505 216_15 94.1 0.1 GCTaaacactataagGAT 27505 217_1 25 106.4 4.9CCcgtaagcatttgagAA 27831 217_1 5 74.6 2.5 CCcgtaagcatttgagAA 27831 218_125 47.0 1.4 AGccaatatgcgacagtaAC 28146 218_1 5 78.1 2.2AGccaatatgcgacagtaAC 28146 219_1 25 42.8 0.3 TAAccaaaacaatcagtGTC 28777219_1 5 62.6 1.1 TAAccaaaacaatcagtGTC 28777 220_1 25 81.1 20.6AACagggaacaggagTTA 29195 220_1 5 78.4 3.3 AACagggaacaggagTTA 29195 221_125 8.6 1.3 ATttatcaacttcCACC 29906 221_1 5 44.4 7.4 ATttatcaacttcCACC29906 222_1 25 40.0 1.7 CGTTtaagaccaggcAC 30020 222_1 5 79.5 8.0CGTTtaagaccaggcAC 30020 223_1 25 45.9 6.2 ACAaaggaactcaggaagAG 30424223_1 5 84.1 9.3 ACAaaggaactcaggaagAG 30424 224_1 25 3.8 0.0TCACagacaagcaccAA 31150 224_1 5 35.8 0.3 TCACagacaagcaccAA 31150 225_125 26.7 1.0 TACTttttaaaacacgtaGG 31329 225_1 5 90.9 3.5TACTttttaaaacacgtaGG 31329 226_1 25 47.9 0.4 GCACaatcacaaagaccAA 31531226_1 5 65.0 9.4 GCACaatcacaaagaccAA 31531 227_1 25 25.4 0.6TCagtaaagaacagaGGC 31820 227_1 5 64.2 1.5 TCagtaaagaacagaGGC 31820 228_125 45.1 2.0 CAtatttccaccacacaAG 32222 228_1 5 66.9 5.1CAtatttccaccacacaAG 32222 229_1 25 7.8 0.9 AGtaaaccactgtCCA 32601 229_15 34.2 2.7 AGtaaaccactgtCCA 32601 230_1 25 23.4 1.2 TCctctttggcgatATA33337 230_1 5 64.4 3.6 TCctctttggcgatATA 33337 231_1 25 50.2 4.4CAtaaatacccctgaaTAC 33986 231_1 5 96.2 6.9 CAtaaatacccctgaaTAC 33986232_1 25 7.2 0.8 CGATtttatcaccaaCA 34175 232_1 5 20.8 2.3CGATtttatcaccaaCA 34175 233_1 25 42.1 1.5 ACaatcaggttaagtgtgGA 34771233_1 5 47.4 7.5 ACaatcaggttaagtgtgGA 34771 234_1 25 36.5 14.4GAAgccaaagactacCA 35096 234_1 5 67.8 4.2 GAAgccaaagactacCA 35096 235_125 63.4 6.2 TGgtagggactgaatttTAA 35850 235_1 5 80.3 0.4TGgtagggactgaatttTAA 35850 236_1 25 93.2 3.6 CGgtagtcaatcACC 36584 236_15 91.2 12.4 CGgtagtcaatcACC 36584 237_1 25 65.0 13.9CTatcaaaattatttcACCT 36886 237_1 5 71.2 6.8 CTatcaaaattatttcACCT 36886238_1 25 96.9 0.6 ACGaaaatttagcatccTAA 37041 238_1 5 75.9 13.5ACGaaaatttagcatccTAA 37041 239_1 25 9.5 1.7 TGgtaaacactggGC 38059 239_15 37.9 1.5 TGgtaaacactggGC 38059 240_1 25 52.4 0.7 GATtgttggttgtcATG38173 240_1 5 63.9 3.4 GATtgttggttgtcATG 38173 241_1 25 38.8 0.7AATtataccccacatttCA 38806 241_1 5 108.3 7.0 AATtataccccacatttCA 38806242_1 25 71.1 1.1 TGcaattagacacgtTACG 39004 242_1 5 83.9 3.6TGcaattagacacgtTACG 39004 243_1 25 42.7 2.6 GGCAaccaattaaacTA 40226243_1 5 71.3 1.9 GGCAaccaattaaacTA 40226 244_1 25 4.6 0.9CAGTtttatgctaatCA 40272 244_1 5 10.6 1.7 CAGTtttatgctaatCA 40272 245_125 79.2 7.3 GCaaaggaggagcggaatAA 40707 245_1 5 81.1 7.2GCaaaggaggagcggaatAA 40707 246_1 25 116.1 0.8 AAacagaagtaagaagGTCC 41156246_1 5 105.7 10.4 AAacagaagtaagaagGTCC 41156 247_1 25 47.5 5.3TCcacttatccataGAAA 41477 248_1 25 50.6 3.5 GCtttgacgaacaggaAAT 42282248_1 5 72.3 0.8 GCtttgacgaacaggaAAT 42282 249_1 25 53.7 2.7TTCCgaccaaaagaaaagAC 42632 249_1 5 115.4 21.3 TTCCgaccaaaagaaaagAC 42632250_1 25 51.9 0.3 GCGacacgatccgttaAA 43104 250_1 5 50.9 3.1GCGacacgatccgttaAA 43104 251_1 25 75.9 2.7 CCCCgttttaaaaAC 43334 251_1 5111.5 3.0 CCCCgttttaaaaAC 43334

EXAMPLE 6 Effect on HBV Infected dHepaRG Cells using Selected AntisenseOligonucleotides Targeting PAPD5 or PAPD7 Delivered to the Cells byGymnosis

A selection of the most efficacious oligonucleotides from Example 4 and5 were selected to test their effect on HBV propagation parameters inHBV infected dHepaRG cells.

HBV infected dHepaRG cells (described in the Materials and Methodssection, HBV infection of dHepaRG cells) were cultured in 96-wellplates. One day post HBV infection 20 μM of oligonucleotide wasdelivered to the cells using unassisted uptake (gymnosis). A total of 40oligonucleotides were tested, 20 targeting PAPD5 and 20 targeting PAPD7.The experiments were conducted in triplicate, with PBS controls. Theoligonucleotide treatment was repeated at day 4 and 7 including mediumreplacement (this differs from the every 2 day replacement described inthe Materials and Method section).

At day 11 post-infection, supernatants were harvested and HBsAg andHBeAg levels were assessed using the CLIA ELISA assay (see Materials andMethods, HBV antigen measurements). Cell viability was measured asdescribed in the Materials and Method section, Cell viability. mRNA wasextracted from the cells using a MagNA Pure robot and the MagNA Pure 96Cellular RNA Large Volume Kit (Roche, #05467535001) according to themanufacturer's protocol. HBV mRNA and PAPD5 or PAPD7 mRNA was quantifiedin technical duplicate by qPCR using a QuantStudio 12K Flex (AppliedBiosystems), the TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems,#4392938), Human ACTB endogenous control (Applied Biosystems,#4310881E). Taqman reagents were used together with the followingcommercial ThermoFisher Sceintific primers (HBV Pa03453406_s1; PAPD5Hs00900790_m1; and PAPD7 Hs00173159_m1). The mRNA expression wasanalyzed using the comparative cycle threshold 2-ΔΔCt method normalizedto the reference gene ACTB (Thermo Fisher Sceintific 4310881E) and toPBS treated cells.

The effects of the oligonucleotide treatment on the PAPD5 or PAPD7 mRNAas well as the effect on the HBV propagation parameter HBsAg are shownin Table 16 and Table 17.

TABLE 16  in vitro effect of PAPD5 targeting compounds ontarget mRNA and HBsAg (average of 3) Start % mRNA on inhi- % HBsAg SEQCMP bition Inhibition ID ID NO Avg sd Avg sd Compound (CMP) NO 10  15_179.1 8.5 −9.7 5.7 AATagcaagtagaggaGAG 3059  18_1 94.0 1.6 −5.3 6.0GTaatacctctcacATTC 5928  23_1 95.9 0.8 45.8 3.9 AGgcttccattttattGAA 8625 25_1 98.5 0.3 4.4 7.1 GTAttcttattcttgCT 10254  26_1 97.5 0.4 20.9 7.0ATTAttcccacagtaaGA 10881  30_1 89.2 3.5 −13.2 1.9 CCAgagtaaacaaaTCC12718  32_1 93.6 1.1 0.8 12.0 TTTggtaattcttttttT 14297 AG  39_1 72.1 5.6−19.4 9.9 GTtaggattttgcTATG 18299  54_1 91.0 1.6 5.2 4.9TTCAacctttactgcAT 27813  56_1 95.6 1.0 5.5 6.6 CAgtaggaatgtggCTT 28718 58_1 82.2 2.6 −18.6 17.5 TCactgttaaaccTCAC 29902  65_1 51.1 6.0 −36.815.4 TTCAcaacaggtaaagGG 33593  80_1 89.4 1.6 9.4 9.9 CAAaggttgttgtacTCT44220  88_1 22.6 10.7 −20.1 13.8 AGCTtaaagaagaacggTT 49688  92_1 93.11.7 −8.1 6.5 GTatctttctgtacTATT 52461  93_1 67.3 5.5 −14.8 2.5GTCAttctactaacaaaCG 53305 111_1 72.0 4.6 −27.6 8.7 ATggttttgtggagaAGG63000 115_1 61.7 6.9 25.8 9.4 GATGgtgacaaattattCT 65894 116_1 97.2 0.711.2 9.5 TGcttttgggaatCTTT 66650 118_1 44.4 15.0 −24.2 24.1GTAcaaggacaaagtaACC 68732

TABLE 17  in vitro effect of PAPD7 targeting compounds ontarget mRNA and HBsAg Average of 3). Start % mRNA on inhi- % HBsAg SEQCMP bition Inhibition ID ID NO Avg sd Avg sd Compound (CMP) NO 11 153_187.5 2.1 −32.3 9.5 CAgtagtaaccacCAAG 7447 155_1 83.6 3.0 −35.8 13.5ACttagtaatagcaGCA 8074 168_1 96.0 1.0 −45.2 9.5 AAtgcttaatttttctCT 11084CT 171_1 96.3 0.8 −34.4 15.3 CAGcataattgttttcTTT 12330 172_1 96.0 0.8−45.0 12.5 ATGTcattatgttttagTT 12634 174_1 87.0 1.5 −28.8 4.8CGgtaagggttcggTG 13126 183_1 96.3 0.7 −31.7 11.6 CATTgcttttataatccTA15816 184_1 85.3 1.9 −46.6 11.2 CTTTttaaggacaggaGG 15988 188_1 89.2 0.8−34.8 20.7 GCAaatgtaagcctttTT 17303 190_1 92.0 0.9 −31.5 15.7AAgagtgggttgtaAGC 17963 191_1 93.4 0.4 −36.2 21.8 TAgtgaaaatatttggAG18101 TT 194_1 65.4 4.0 −37.3 17.4 GTAggtaaagggcaaagg 19007 AA 195_185.9 3.8 −39.2 14.6 GTgaattaaagccaAAGC 19252 197_1 94.0 1.3 −24.9 15.5GAggttttttttagtgAA 19722 TT 221_1 99.1 0.3 −36.4 4.7 ATttatcaacttcCACC29906 224_1 97.7 0.6 −22.1 16.0 TCACagacaagcaccAA 31150 229_1 96.6 0.760.5 4.6 AGtaaaccactgtCCA 32601 232_1 95.5 1.5 −48.3 31.0CGATtttatcaccaaCA 34175 239_1 93.1 1.3 −31.6 3.7 TGgtaaacactggGC 38059244_1 98.7 0.3 −26.9 8.5 CAGTtttatgctaatCA 40272

From these data it can be seen that the inhibition of the PAPD5 mRNA(table 16) and PAPD7 mRNA (table 17) was very efficient for most of theoligonucleotide compounds.

The observed effect of PAPD5 and PAPD7 mRNA reduction on HBsAg levels ishowever less pronounced even in cells treated for 11 days afterun-assisted delivery of oligonucleotide. Here only PAPD5 targeting CMPID NO: 23_1, 26_1 and 115_1 showed clear HBsAg inhibition and of thePAPD7 targeting compounds only CPM ID NO: 229_1 showed clear HBsAginhibition. Without being bound by theory this could be due to a slowonset of the effect caused by the target knock down on the HBsAginhibition in the present assay and thus, for some compounds, HBsAginhibition would not be seen unless assayed at a later time point.

EXAMPLE 7 Effect on HBV Infected dHepaRG Cells using a CombinedPreparation of a PAPD5 and a PAPD7 Targeting Antisense OligonucleotideDelivered to the Cells by Gymnosis.

In Example 2 it was observed that mixing a pool of PAPD7 targetingsiRNA's with a pool of PAPD5 targeting siRNA's resulted in a synergisticanti-HBV activity. The present example sets out to investigate whether asimilar synergy can be observed when combining two individual singlestranded antisense oligonucleotides, where one targets PAPD5 and onetargets PAPD7.

The experiment was conducted as described in Example 6 with the changethat instead of adding individual oligonucleotides, a combination of twooligonucleotides were added to the cells, such that 20 μM of oneoligonucleotide targeting PAPD5 was added together with 20 μM of asecond oligonucleotide targeting PAPD7. Only HBsAg was measured for thecombinations.

The combination of oligonucleotides can be seen in table 18, includingthe results on the HBV propagation parameters, HBsAg inhibition.

TABLE 18 in vitro effect combinations of PAPD5 and PAPD7 targetingcompounds on HBsAg (average of 3). The HBsAg inhibition results fromtable 16 and 17 on the individual compounds are also included in thistable for ease of comparing the individual treatment with thecombination treatment. % HBsAg % HBsAg % HBsAg inhibition inhibitioninhibition CMP ID NO combination PAPD5 PAPD7 combination Avg sd Avg sdAvg sd 15_1 + 184_1 −14.9 13.0 −9.7 5.7 −46.6 11.2 15_1 + 221_1 −8.1 2.9−9.7 5.7 −36.4 4.7 18_1 + 184_1 −8.0 1.8 −5.3 6.0 −46.6 11.2 18_1 +221_1 22.0 10.8 −5.3 6.0 −36.4 4.7 23_1 + 172_1 48.4 6.3 45.8 3.9 −45.012.5 23_1 + 188_1 47.2 14.3 45.8 3.9 −34.8 20.7 25_1 + 174_1 31.3 7.84.4 7.1 −28.8 4.8 25_1 + 183_1 58.5 9.9 4.4 7.1 −31.7 11.6 26_1 + 174_18.4 2.6 20.9 7.0 −28.8 4.8 26_1 + 183_1 38.5 11.2 20.9 7.0 −31.7 11.630_1 + 172_1 −19.1 11.2 −13.2 1.9 −45.0 12.5 30_1 + 188_1 −8.6 5.0 −13.21.9 −34.8 20.7 32_1 + 155_1 21.0 1.2 0.8 12.0 −35.8 13.5 32_1 + 195_18.3 4.7 0.8 12.0 −39.2 14.6 39_1 + 224_1 −10.7 10.0 −19.4 9.9 −22.1 16.039_1 + 229_1 48.3 4.8 −19.4 9.9 60.5 4.6 54_1 + 190_1 8.3 9.1 5.2 4.9−31.5 15.7 54_1 + 232_1 29.4 2.4 5.2 4.9 −48.3 31.0 56_1 + 153_1 45.09.5 5.5 6.6 −32.3 9.5 56_1 + 244_1 60.5 5.9 5.5 6.6 −26.9 8.5 58_1 +171_1 −8.0 17.8 −18.6 17.5 −34.4 15.3 58_1 + 239_1 −14.2 13.0 −18.6 17.5−31.6 3.7 65_1 + 171_1 −21.2 4.5 −36.8 15.4 −34.4 15.3 65_1 + 239_1−26.4 2.3 −36.8 15.4 −31.6 3.7 80_1 + 153_1 53.6 5.1 9.4 9.9 −32.3 9.580_1 + 244_1 65.4 7.3 9.4 9.9 −26.9 8.5 88_1 + 168_1 −16.4 7.9 −20.113.8 −45.2 9.5 88_1 + 197_1 −9.7 9.0 −20.1 13.8 −24.9 15.5 92_1 + 190_134.6 7.2 −8.1 6.5 −31.5 15.7 92_1 + 232_1 23.4 11.6 −8.1 6.5 −48.3 31.093_1 + 224_1 12.8 5.6 −14.8 2.5 −22.1 16.0 93_1 + 229_1 27.7 10.3 −14.82.5 60.5 4.6 111_1 + 191_1  −20.0 7.0 −27.6 8.7 −36.2 21.8 111_1 +194_1  −22.2 5.6 −27.6 8.7 −37.3 17.4 115_1 + 191_1  4.8 5.4 25.8 9.4−36.2 21.8 115_1 + 194_1  −6.7 13.3 25.8 9.4 −37.3 17.4 116_1 + 155_1 39.2 2.2 11.2 9.5 −35.8 13.5 116_1 + 195_1  22.9 9.5 11.2 9.5 −39.2 14.6118_1 + 168_1  −9.8 11.5 −24.2 24.1 −45.2 9.5 118_1 + 197_1  4.6 10.5−24.2 24.1 −24.9 15.5

The results are also summarized in FIG. 10 . Of the 40 combinationstested above 18 resulted in an apparent synergistic effect on the HBsAginhibition. 7 of these synergistic combinations are witholigonucleotides that individually did not produce any effect on HBsAginhibition in the current assay. From this experiment it can beconcluded that the combination of PAPD5 and a PAPD7 targetingoligonucleotides have a high likelihood of producing a synergisticeffect on HBsAg inhibition.

EXAMPLE 8 Repeat of Selected Combinations from Example 7

The experiment in example 7 was repeated with the oligonucleotidecombinations indicated in table 19.

TABLE 19 in vitro effect combinations of PAPD5 and PAPD7 targetingcompounds on HBsAg (average of 3). The HBsAg inhibition results fromtable 16 and 17 on the individual compounds are also included in thistable for ease of comparing the individual treatment with thecombination treatment. % HBsAg % HBsAg % HBsAg inhibition inhibitioninhibition CMP ID NO combination PAPD5 PAPD7 combination Avg sd Avg sdAvg sd 23_1 + 172_1 57.4 13.2 45.8 3.9 −45.0 12.5 23_1 + 188_1 63.6 8.045.8 3.9 −34.8 20.7 25_1 + 174_1 36.2 14.5 4.4 7.1 −28.8 4.8 25_1 +183_1 80.3 1.3 4.4 7.1 −31.7 11.6 39_1 + 229_1 33.9 18.5 −19.4 9.9 60.54.6 54_1 + 232_1 56.0 8.5 5.2 4.9 −48.3 31.0 56_1 + 153_1 55.4 6.5 5.56.6 −32.3 9.5 80_1 + 153_1 53.2 9.3 9.4 9.9 −32.3 9.5 80_1 + 244_1 74.35.1 9.4 9.9 −26.9 8.5 92_1 + 190_1 27.8 9.9 −8.1 6.5 −31.5 15.7 116_1 +155_1  31.6 1.1 11.2 9.5 −35.8 13.5

From these data it can be seen that the synergistic effect observed inexample 7 is repeatable.

EXAMPLE 9 Effect on HBV Infected dHepaRG Cells using Selected AntisenseOligonucleotides Targeting PAPD5 or PAPD7 Delivered to the Cells byTransfection

In the following experiment it was investigated whether similarsynergistic results could be achieved using transfection of theoligonucleotides into HBV infected dHepaRG cells instead of unassisteddelivery.

Twelve PAPD5 targeting oligonucleotides (table 20) and thirteen PAPD7targeting oligonucleotides (table 21) were tested individually using thetransfection assay described here. Oligonucleotides were transfected ina 96-well plate format at a final concentration of 500 nM per well indifferentiated HepaRG cells one day post-infection with HBV (describedin the Materials and Methods section, HBV infection of dHepaRG cells).Prior to transfection, medium was replaced with 100 uLpenicillin/streptomycin free complete differentiation medium. For singleoligonucleotide treatment, oligonucleotide was diluted inOpti-MEM+Glutamax-I reduced serum medium (Gibco, #51985) and incubatedat a ratio 1:1 with Lipofectamine RNAiMax (Invitrogen, #56532) for 5minutes at room temperature according to the manufacturer'sinstructions. From this LNA-transfection reagent mixture, 20 μl was thenadded on top of the cells. After 3 days, medium was replaced with acomplete differentiation medium. At day 5 post-transfection, HBsAg wasmeasured as described in the materials and method section, PAPD5 andPAPD7 mRNA was measured as described in Example 6.

In addition some of the PAPD5 and PAPD7 oligonucleotides were tested incombination (table 22). The oligonucleotides were co-transfected intothe HBV infected HepaRG cells using the same protocol as singletreatment with 500 nM of each oligonucleotide in the finalconcentration.

TABLE 20  in vitro effect of PAPD5 targeting compoundson target mRNA and HBsAg. Start % mRNA on CMP inhi- % HBsAg SEQ IDbition Inhibition ID NO Avg sd Avg sd Compound (CMP) NO 10 23_1 70.3 4.089.2 8.7 AGgcttccattttatt 8625 GAA 25_1 81.0 2.9 3.9 12.6GTAttcttattcttgCT 10254 26_1 85.6 1.8 52.7 14.0 ATTAttcccacagtaaGA 1088139_1 69.3 5.8 14.3 10.0 GTtaggattttgcTATG 18299 54_1 75.6 3.6 31.6 16.5TTCAacctttactgcAT 27813 56_1 90.7 1.4 67.4 5.3 CAgtaggaatgtggCTT 2871858_1 39.3 11.1 -4.1 14.6 TCactgttaaaccTCAC 29902 65_1 81.1 4.8 32.3 34.5TTCAcaacaggtaaagGG 33593 80_1 90.9 1.7 33.2 8.7 CAAaggttgttgtacTCT 4422092_1 80.5 1.9 15.3 19.1 GTatctttctgtacTATT 52461 93_1 72.2 4.3 37.3 12.5GTCAttctactaacaaa 53305 CG 116_1  81.6 4.8 10.8 10.3 TGcttttgggaatCTTT66650

TABLE 21  in vitro effect of PAPD7 targeting compounds ontarget mRNA and HBsAg % mRNA Start inhi- % HBsAg on CMP bitionInhibition SEQ ID ID NO Avg sd Avg sd Compound (CMP) NO 11 153_1 67.08.3 −17.8 9.4 CAgtagtaaccacCAAG 7447 155_1 86.3 4.2 0.1 31.9ACttagtaatagcaGCA 8074 171_1 66.8 13.2 −5.8 6.6 CAGcataattgttttcTTT12330 172_1 94.5 1.2 12.1 6.8 ATGTcattatgttttagTT 12634 174_1 58.6 7.3−6.8 21.9 CGgtaagggttcggTG 13126 183_1 74.1 5.0 6.7 23.6CATTgcttttataatccTA 15816 188_1 72.8 4.7 26.9 4.6 GCAaatgtaagcctttTT17303 190_1 75.2 4.9 87.7 1.0 AAgagtgggttgtaAGC 17963 224_1 72.6 4.4 4.324.1 TCACagacaagcaccAA 31150 229_1 48.0 6.4 27.8 24.9 AGtaaaccactgtCCA32601 232_1 64.0 4.0 −15.1 38.3 CGATtttatcaccaaCA 34175 239_1 91.9 3.788.0 7.0 TGgtaaacactggGC 38059 244_1 70.0 3.6 −12.9 11.3CAGTtttatgctaatCA 40272

TABLE 22 in vitro effect of combinations of PAPD5 and PAPD7 targetingcompounds. The HBsAg inhibition results from table 20 and 21 on theindividual compounds are also included in this table for ease ofcomparing the individual treatment with the combination treatment %HBsAg inhibition CMP ID % HBsAg CMP ID % HBsAg CMP ID NO combination NOinhibition NO inhibition combination Avg sd PAPD5 Avg sd PAPD7 Avg sd23_1 + 172_1 44.1 6.9 23_1 89.2 8.7 172_1 12.1 6.8 23_1 + 188_1 76.612.0 23_1 89.2 8.7 188_1 26.9 4.6 25_1 + 174_1 33.0 22.1 25_1 3.9 12.6174_1 −6.8 21.9 25_1 + 183_1 75.3 7.8 25_1 3.9 12.6 183_1 6.7 23.639_1 + 229_1 77.4 13.3 39_1 14.3 10.0 229_1 27.8 24.9 54_1 + 232_1 79.96.5 54_1 31.6 16.5 232_1 −15.1 38.3 56_1 + 153_1 95.6 1.2 56_1 67.4 5.3153_1 −17.8 9.4 56_1 + 244_1 89.2 4.8 56_1 67.4 5.3 244_1 −12.9 11.380_1 + 153_1 85.5 2.3 80_1 33.2 8.7 153_1 −17.8 9.4 80_1 + 244_1 82.86.7 80_1 33.2 8.7 244_1 −12.9 11.3 92_1 + 190_1 90.5 1.9 92_1 15.3 19.1190_1 87.7 1.0 116_1 + 155_1  87.5 2.5 116_1  10.8 10.3 155_1 0.1 31.9

From these data it can be seen that when using a transfection assay 6out of the 12 oligonucleotides targeting PAPD5 have a clear effect onHBsAg inhibition, which is considerably more than observed with thegymnotic assay in example 6. Also the number of PAPD7 targetingcompounds that have an effect on the HBsAg inhibition has been increasedusing the transfection assay such that 4 out of 12 compounds have aclear effect on HBsAg inhibition. The data from the combinations aresummarized in table 22 and FIG. 11 . From these data it can be seen that9 of the 12 tested combinations produce an apparent synergistic effecton the HBsAg inhibition.

1. A composition comprising a nucleic acid molecule for use in thetreatment and/or prevention of Hepatitis B virus infection, wherein saidnucleic acid molecule inhibits expression and/or activity of PAPassociated domain containing 5 (PAPD5).
 2. The composition of claim 1,wherein said composition is a combined preparation comprising: a. anucleic acid molecule which inhibits expression and/or activity PAPD5;and b. a nucleic acid molecule which inhibits expression and/or activityof PAP associated domain containing 7 (PAPD7).
 3. The composition ofclaim 1, wherein the nucleic acid molecules are independently selectedfrom the group consisting of: a. a single stranded antisenseoligonucleotide; b. a siRNA molecule; c. a shRNA molecule; and d. agenome editing machinery, comprising: i. a site-specific DNA nuclease ora polynucleotide encoding a site-specific DNA nuclease; and ii. a guideRNA or a polynucleotide encoding a guide RNA.
 4. The composition ofclaim 2, wherein the nucleic acid molecules are selected from: a. asingle stranded antisense oligonucleotide comprising a contiguousnucleotide sequence of 10 to 30 nucleotides in length with at least 80%complementarity to PAPD5 target nucleic acid and which capable ofreducing expression of PAPD5; and b. a single stranded antisenseoligonucleotide comprising a contiguous nucleotide sequence of 10 to 30nucleotides in length with at least 80% complementarity to a PAPD7target nucleic acid and which is capable of reducing expression ofPAPD7.
 5. The composition of claim 1, wherein the composition reducessecretion of HBsAg, HBeAg and/or inhibits production of intracellularHBV mRNA or HBV DNA.
 6. The composition of claim 1, wherein thecomposition inhibits development of chronic HBV infection and/or reducesthe infectiousness of a HBV infected person.
 7. A method for identifyinga compound or composition that prevents, ameliorates and/or inhibits ahepatitis B virus (HBV) infection, comprising: a. contacting a testcompound or composition with a cell expressing PAPD5 and/or PAPD7; b.measuring the expression and/or activity of PAPD5 and/or PAPD7 in thepresence and absence of said test compound or composition; and c.identifying a compound or composition that reduces the expression and/oractivity of PAPD5 and/or PAPD7 as a compound that prevents, amelioratesand/or inhibits a HBV infection.
 8. The method of claim 7, wherein thetest compound is a library of nucleic acid molecules and step c)identifies nucleic acid molecules that reduce PAPD5 or PAPD7 mRNAexpression by at least 60%.
 9. The method of claim 7, wherein the testcomposition is a combined preparation of a nucleic add molecule capableof reducing PAPD5 and a nucleic add molecule capable of reducing PAPD7.10. The method of claim 7, wherein the compound that inhibitspropagation of HBV inhibits secretion of HBV surface antigen (HBsAg),and/or inhibits secretion of HBV envelope antigen (HBeAg), and/orinhibits production of intracellular HBV mRNA.
 11. A single strandedantisense oligonucleotide which comprises a contiguous nucleotidesequence of 10 to 30 nucleotides in length wherein the contiguousnucleotide sequence is at least 80% complementarity to a PAPD5 targetnucleic add and the antisense oligonucleotide is capable of reducingexpression of PAPD5.
 12. A nucleic add molecule which comprises acontiguous nucleotide sequence of 10 to 30 nucleotides in length whereinthe contiguous nucleotide sequence is at least 80% complementarity to aPAPD7 target nucleic add and the nucleic add molecule is capable ofreducing expression of PAPD7.
 13. The nucleic add molecule of claim 12,wherein the nucleic add molecule is a single stranded antisenseoligonucleotide.
 14. The antisense oligonucleotide or nucleic addmolecule of claim 11, comprising one or more 2′ sugar modifiednucleoside(s).
 15. The antisense oligonucleotide or nucleic add moleculeof claim 14, wherein the one or more 2′ sugar modified nucleoside isindependently selected from the group consisting of 2′-0-alkyl-RNA,2′-0-methyl-RNA, 2′-alkoxy-RNA, 2′-0-methoxyethyl-RNA, 2′-amino-DNA,2′-fluoro-DNA, arabino nucleic add (ANA), 2′-fluoro-ANA and LNAnucleosides.
 16. The antisense oligonucleotide or nucleic add moleculeof claim 14, wherein the one or more 2′ sugar modified nucleoside is aLNA nucleoside.
 17. The antisense oligonucleotide or nucleic acidmolecule of claim 11, wherein the internucleoside linkages within thecontiguous nucleotide sequence are phosphorothioate internucleosidelinkages.
 18. The antisense oligonucleotide of claim 11, wherein theoligonucleotide is a gapmer of formula 5′-F-G-F′-3′, where region F andF′ independently comprise 1-7 2′ sugar modified nucleosides and G is aregion between 6 and 16 nucleosides which are capable of recruitingRNaseH.
 19. A conjugate comprising the antisense oligonucleotideaccording to claim 11, and at least one conjugate moiety covalentlyattached to said oligonucleotide.
 20. A combined preparation comprisinga. a nucleic acid molecule which inhibits expression and/or activity ofPAPD5; and b. a nucleic acid molecule which inhibits expression and/oractivity of PAPD7.
 21. The combined preparation of claim 20, wherein thenucleic acid molecule is a single stranded antisense oligonucleotidewhich comprises or consists of a contiguous nucleotide sequence of 10 to30 nucleotides in length wherein the contiguous nucleotide sequence isat least 80% complementarity to a PAPD5 target nucleic acid and theantisense oligonucleotide is capable of reducing expression of PAPD5.22. A pharmaceutical composition comprising the antisenseoligonucleotide or nucleic acid molecule according to claim 11.